103 results on '"Katarzyna Bebenek"'
Search Results
2. Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break
- Author
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Andrea M. Kaminski, John M. Pryor, Dale A. Ramsden, Thomas A. Kunkel, Lars C. Pedersen, and Katarzyna Bebenek
- Subjects
Science - Abstract
Polymerase μ (Polμ) participates in the repair of DNA double-strand breaks (DSBs) via the nonhomologous end-joining (NHEJ) pathway. Here, the authors determine the crystal structure of a pre-catalytic ternary complex of human Polμ with a bound DSB substrate and they obtain further mechanistic insights by allowing the insertion reaction to proceed in crystallo, which enabled them to determine a Polμ structure with incomplete incorporation and the structure of the post-catalytic nicked state.
- Published
- 2020
- Full Text
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3. Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis
- Author
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Andrea M. Kaminski, Percy P. Tumbale, Matthew J. Schellenberg, R. Scott Williams, Jason G. Williams, Thomas A. Kunkel, Lars C. Pedersen, and Katarzyna Bebenek
- Subjects
Science - Abstract
DNA Ligase IV (LigIV) catalyzes nick sealing of DNA double-strand break substrates during non-homologous end-joining. Here the authors present the crystal structures of two human LigIV DNA-bound catalytic states, which provide insights into its catalytic mechanism and the molecular basis of LIG4 syndrome causing disease mutations.
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- 2018
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- View/download PDF
4. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action
- Author
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Joonas A. Jamsen, William A. Beard, Lars C. Pedersen, David D. Shock, Andrea F. Moon, Juno M. Krahn, Katarzyna Bebenek, Thomas A. Kunkel, and Samuel H. Wilson
- Subjects
Science - Abstract
DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.
- Published
- 2017
- Full Text
- View/download PDF
5. Characterization of a natural mutator variant of human DNA polymerase lambda which promotes chromosomal instability by compromising NHEJ.
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Gloria Terrados, Jean-Pascal Capp, Yvan Canitrot, Miguel García-Díaz, Katarzyna Bebenek, Tomas Kirchhoff, Alberto Villanueva, François Boudsocq, Valérie Bergoglio, Christophe Cazaux, Thomas A Kunkel, Jean-Sébastien Hoffmann, and Luis Blanco
- Subjects
Medicine ,Science - Abstract
BACKGROUND:DNA polymerase lambda (Pollambda) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB). PRINCIPAL FINDINGS:Here, we described a novel natural allelic variant of human Pollambda (hPollambda) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Pollambda variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPollambda variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPollambda. CONCLUSIONS:The expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.
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- 2009
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6. Correction: Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ.
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Gloria Terrados, Jean-Pascal Capp, Yvan Canitrot, Miguel García-Díaz, Katarzyna Bebenek, Tomas Kirchhoff, Alberto Villanueva, François Boudsocq, Valérie Bergoglio, Christophe Cazaux, Thomas A. Kunkel, Jean-Sébastien Hoffmann, and Luis Blanco
- Subjects
Medicine ,Science - Published
- 2009
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7. Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Family X DNA Polymerases
- Author
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Andrea M. Kaminski, Thomas A. Kunkel, Lars C. Pedersen, and Katarzyna Bebenek
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DNA Replication ,8-oxo-guanine (8OG) ,DNA Repair ,Family X polymerases ,Review ,DNA ,DNA-Directed DNA Polymerase ,QH426-470 ,base excision repair (BER) ,nonhomologous end-joining (NHEJ) ,oxidized base damage ,8-Hydroxy-2'-Deoxyguanosine ,Catalytic Domain ,Genetics ,Humans ,Genetics (clinical) - Abstract
8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols β, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles—the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.
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- 2021
8. Analysis of diverse double-strand break synapsis with Polλ reveals basis for unique substrate specificity in nonhomologous end-joining
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Andrea M. Kaminski, Kishore K. Chiruvella, Dale A. Ramsden, Katarzyna Bebenek, Thomas A. Kunkel, and Lars C. Pedersen
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Chromosome Pairing ,Multidisciplinary ,DNA End-Joining Repair ,Synapses ,General Physics and Astronomy ,DNA Breaks, Double-Stranded ,General Chemistry ,Nucleotidyltransferases ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity - Abstract
DNA double-strand breaks (DSBs) threaten genomic stability, since their persistence can lead to loss of critical genetic information, chromosomal translocations or rearrangements, and cell death. DSBs can be repaired through the nonhomologous end-joining pathway (NHEJ), which processes and ligates DNA ends efficiently to prevent or minimize sequence loss. Polymerase λ (Polλ), one of the Family X polymerases, fills sequence gaps of DSB substrates with a strict specificity for a base-paired primer terminus. There is little information regarding Polλ’s approach to engaging such substrates. We used in vitro polymerization and cell-based NHEJ assays to explore the contributions of conserved loop regions toward DSB substrate specificity and utilization. In addition, we present multiple crystal structures of Polλ in synapsis with varying biologically relevant DSB end configurations, revealing how key structural features and hydrogen bonding networks work in concert to stabilize these tenuous, potentially cytotoxic DNA lesions during NHEJ.
- Published
- 2021
9. Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine
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Katarzyna Bebenek, Andrea M. Kaminski, Kishore K. Chiruvella, Lars C. Pedersen, Thomas A. Kunkel, and Dale A. Ramsden
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DNA Replication ,DNA End-Joining Repair ,Ultraviolet Rays ,Stereochemistry ,Guanosine ,DNA-Directed DNA Polymerase ,DNA Ligase ATP ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Structural Biology ,Radiation, Ionizing ,Genetics ,Humans ,Transferase ,DNA Breaks, Double-Stranded ,Nucleotide ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,DNA ligase ,biology ,030302 biochemistry & molecular biology ,Active site ,chemistry ,Mutagenesis ,biology.protein ,Reactive Oxygen Species ,DNA polymerase mu ,DNA ,DNA Damage - Abstract
DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.
- Published
- 2019
10. DNA polymerase mu: An inflexible scaffold for substrate flexibility
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Lars C. Pedersen, Katarzyna Bebenek, Andrea M. Kaminski, and Thomas A. Kunkel
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DNA End-Joining Repair ,DNA polymerase ,DNA-Directed DNA Polymerase ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,DNA Ligase ATP ,0302 clinical medicine ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,DNA ligase ,biology ,fungi ,Substrate (chemistry) ,Cell Biology ,DNA ,Non-homologous end joining ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Biophysics ,DNA polymerase mu ,Recombination - Abstract
DNA polymerase μ is a Family X member that participates in repair of DNA double strand breaks (DSBs) by non-homologous end joining. Its role is to fill short gaps arising as intermediates in the process of V(D)J recombination and during processing of accidental double strand breaks. Pol μ is the only known template-dependent polymerase that can repair non-complementary DSBs with unpaired 3´primer termini. Here we review the unique properties of Pol μ that allow it to productively engage such a highly unstable substrate to generate a nick that can be sealed by DNA Ligase IV.
- Published
- 2020
11. Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break
- Author
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Katarzyna Bebenek, Andrea M. Kaminski, John M. Pryor, Thomas A. Kunkel, Dale A. Ramsden, and Lars C. Pedersen
- Subjects
Models, Molecular ,0301 basic medicine ,DNA End-Joining Repair ,DNA Repair ,Protein Conformation ,DNA damage ,DNA repair ,Science ,General Physics and Astronomy ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,Article ,General Biochemistry, Genetics and Molecular Biology ,DNA Ligase ATP ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Humans ,DNA Breaks, Double-Stranded ,lcsh:Science ,Polymerase ,X-ray crystallography ,chemistry.chemical_classification ,DNA ligase ,Multidisciplinary ,biology ,Synapsis ,Hydrogen Bonding ,DNA ,General Chemistry ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,chemistry ,Coding strand ,biology.protein ,lcsh:Q ,030217 neurology & neurosurgery ,DNA Damage - Abstract
Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks., Polymerase μ (Polμ) participates in the repair of DNA double-strand breaks (DSBs) via the nonhomologous end-joining (NHEJ) pathway. Here, the authors determine the crystal structure of a pre-catalytic ternary complex of human Polμ with a bound DSB substrate and they obtain further mechanistic insights by allowing the insertion reaction to proceed in crystallo, which enabled them to determine a Polμ structure with incomplete incorporation and the structure of the post-catalytic nicked state.
- Published
- 2020
12. The correlation between serum E-selectin levels and soluble interleukin-2 receptors with relation to disease activity in localized scleroderma
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Natalia Salwowska, Dominika Wcisło-Dziadecka, Karolina Wodok-Wieczorek, Aleksandra Wodok, Ewa Syguła, Ligia Brzezińska-Wcisło, Beata Bergler-Czop, and Katarzyna Bebenek
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medicine.medical_specialty ,lcsh:Internal medicine ,Dermatology ,Disease ,Gastroenterology ,Scleroderma ,Serology ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,Fibrosis ,Statistical significance ,Internal medicine ,lcsh:Dermatology ,Immunology and Allergy ,Medicine ,Localized Scleroderma ,lcsh:RC31-1245 ,030203 arthritis & rheumatology ,Original Paper ,medicine.diagnostic_test ,business.industry ,selectin ,E-selectin ,lcsh:RL1-803 ,medicine.disease ,Connective tissue disease ,connective tissue disease ,interleukin-2 receptors ,Skin biopsy ,business ,localized scleroderma - Abstract
Introduction Scleroderma is a chronic connective tissue disease resulting in fibrosis. Aim The aim of the study was to determine the connection between sE-selectin and sIL-2R and the severity of skin lesions in various subtypes of LoS. Evaluation of disease severity, the location of skin lesions, the duration of symptoms and disease activity were assessed in relation to the three different LoS subtypes in patients with localized scleroderma. Material and methods The study included 42 patients with localized scleroderma and the control group consisted of 41 healthy subjects. All patients in the LoS study group had a confirmed diagnosis via skin biopsy and underwent serology testing for sE-selectin and sIL-2R concentrations by enzyme-linked immunosorbent assay (ELISA). Results Significantly higher levels of sE-selectin and sIL-2R were observed in the LoS study group when compared with the control group (p < 0.001). The analysis showed a result close to statistical significance (p = 0.058) between sE-selectin concentration during the time of active disease in the LoS study group. The highest concentrations of sE-selectin and sIL-2R were observed in patients with the generalized subtype of LoS. A positive, statistically significant, curvilinear relationship was shown amid the modified Localized Skin Severity Index (mLoSSI) and sE-selectin and sIL-2R concentrations in the LoS study group. Conclusions Concentrations of the circulating form of sE-selectin appear to be an adequate marker of the endothelial function, positively correlating with the severity of the disease. The proven correlation of sIL-2R concentrations with the severity of the disease indicates that it is a valuable prognostic factor for predicting the impending course of the disease.
- Published
- 2018
13. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action
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Lars C. Pedersen, Joonas A. Jamsen, David D. Shock, Andrea F. Moon, Samuel H. Wilson, William A. Beard, Katarzyna Bebenek, Juno M. Krahn, and Thomas A. Kunkel
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0301 basic medicine ,DNA Replication ,Models, Molecular ,DNA Repair ,DNA polymerase ,DNA repair ,Science ,General Physics and Astronomy ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Catalytic Domain ,DNA Breaks, Double-Stranded ,Polymerase ,Multidisciplinary ,DNA clamp ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Nucleotides ,DNA replication ,General Chemistry ,Processivity ,DNA ,Double Strand Break Repair ,Crystallography ,Kinetics ,030104 developmental biology ,biology.protein - Abstract
DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5′-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance., DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.
- Published
- 2017
14. Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis
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Katarzyna Bebenek, Thomas A. Kunkel, Matthew J. Schellenberg, Percy P. Tumbale, R. Scott Williams, Lars C. Pedersen, Jason Williams, and Andrea M. Kaminski
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0301 basic medicine ,Immunoglobulin gene ,Science ,LIG4 syndrome ,General Physics and Astronomy ,General Biochemistry, Genetics and Molecular Biology ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,DNA Ligase ATP ,Catalytic Domain ,medicine ,Humans ,lcsh:Science ,chemistry.chemical_classification ,DNA ligase ,Multidisciplinary ,Polymorphism, Genetic ,030102 biochemistry & molecular biology ,biology ,Base Sequence ,Chemistry ,Adenine ,Lysine ,Mutagenesis ,Active site ,General Chemistry ,DNA ,medicine.disease ,Cell biology ,030104 developmental biology ,Mutation ,biology.protein ,Nucleic acid ,Biocatalysis ,lcsh:Q ,Function (biology) ,Protein Binding - Abstract
DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct states—an open lysyl-AMP intermediate, and a closed DNA–adenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome., DNA Ligase IV (LigIV) catalyzes nick sealing of DNA double-strand break substrates during non-homologous end-joining. Here the authors present the crystal structures of two human LigIV DNA-bound catalytic states, which provide insights into its catalytic mechanism and the molecular basis of LIG4 syndrome causing disease mutations.
- Published
- 2017
15. Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase Mu
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Thomas A. Kunkel, Katarzyna Bebenek, Andrea F. Moon, John M. Pryor, Lars C. Pedersen, and Dale A. Ramsden
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Models, Molecular ,Protein Conformation, alpha-Helical ,0301 basic medicine ,DNA End-Joining Repair ,Ribonucleotide ,DNA polymerase ,DNA repair ,DNA polymerase II ,Amino Acid Motifs ,Deoxyribonucleotides ,Gene Expression ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,Substrate Specificity ,03 medical and health sciences ,RNTP ,Structural Biology ,Catalytic Domain ,Escherichia coli ,Genetics ,Humans ,Protein Interaction Domains and Motifs ,Cloning, Molecular ,DNA clamp ,Base Sequence ,biology ,DNA ,Ribonucleotides ,Recombinant Proteins ,Kinetics ,030104 developmental biology ,Biochemistry ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,Thermodynamics ,Protein Conformation, beta-Strand ,DNA polymerase mu ,Protein Binding - Abstract
While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol μ with a ribonucleotide bound at the active site. These structures reveal that Pol μ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol μ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol μ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.
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- 2017
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16. DNA Polymerase λ Inactivation by Oxidized Abasic Sites
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Thomas A. Kunkel, Adam J. Stevens, Lirui Guan, Marc M. Greenberg, and Katarzyna Bebenek
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Base Sequence ,biology ,Deoxyribose ,DNA polymerase ,DNA damage ,DNA polymerase beta ,DNA ,Processivity ,Base excision repair ,Biochemistry ,DNA polymerase delta ,Molecular biology ,Butanones ,Article ,Enzyme Activation ,chemistry.chemical_compound ,chemistry ,biology.protein ,Humans ,AP site ,Oxidation-Reduction ,DNA Polymerase beta ,DNA Damage - Abstract
Base excision repair plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ is believed to play a backup role to DNA polymerase β in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA damaging agents, including several antitumor antibiotics, the C4′-oxidized abasic site following Ape1 incision (pC4-AP) and 5′-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single turnover kinetic experiments show that Pol λ excises DOB almost 13-times more slowly than a 5′-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β at the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β and the difference is attributable to a higher KI (33 ± 7 nM). Inactivation of Pol λ’s lyase activity by DOB also prevents the enzyme from carrying out polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. Protein modification involves reaction with released but-2-ene-1,4-dial. When acting on pC4-AP, Pol λ achieves approximately 4 turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA damaging agents that produce them.
- Published
- 2013
17. Altered Ig Hypermutation Pattern and Frequency in Complementary Mouse Models of DNA Polymerase ζ Activity
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Ming-Lang Zhao, Janssen Daly, Danielle L. Watt, Marilyn Diaz, Thomas A. Kunkel, Madhumita Ray, Katarzyna Bebenek, Kathleen Richter, Chuancang Jiang, and W. Glenn McGregor
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DNA polymerase ,Protein subunit ,Immunology ,Gene Rearrangement, B-Lymphocyte, Heavy Chain ,Somatic hypermutation ,Mice, Transgenic ,DNA-Directed DNA Polymerase ,medicine.disease_cause ,Article ,Mice ,Conditional gene knockout ,Homologous chromosome ,medicine ,Animals ,Immunology and Allergy ,Gene Knock-In Techniques ,Mutation frequency ,Gene ,Mice, Knockout ,B-Lymphocytes ,Mutation ,biology ,Molecular biology ,Enzyme Activation ,Mice, Inbred C57BL ,Models, Animal ,biology.protein ,Somatic Hypermutation, Immunoglobulin - Abstract
To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer’s patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer’s patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.
- Published
- 2012
18. The catalytic cycle for ribonucleotide incorporation by human DNA Pol λ
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Lars C. Pedersen, Katarzyna Bebenek, Andrea F. Moon, Rajendrakumar A. Gosavi, and Thomas A. Kunkel
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Models, Molecular ,Ribonucleotide ,DNA polymerase ,DNA polymerase beta ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,RNTP ,Catalytic Domain ,Deoxyribonucleotide Triphosphate ,Genetics ,Humans ,Polymerase ,DNA Polymerase beta ,030304 developmental biology ,0303 health sciences ,biology ,Ribonucleotides ,0104 chemical sciences ,Kinetics ,chemistry ,Biochemistry ,Biophysics ,biology.protein ,Biocatalysis ,RNA ,Primer (molecular biology) ,DNA - Abstract
Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2′-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein–nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and β-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high.
- Published
- 2012
19. Loop 1 modulates the fidelity of DNA polymerase λ
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Rui-Zhe Zhou, Miguel Garcia-Diaz, Lawrence F. Povirk, Katarzyna Bebenek, and Thomas A. Kunkel
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Models, Molecular ,DNA polymerase ,Protein Conformation ,DNA polymerase II ,Deoxyribonucleotides ,Molecular Sequence Data ,Genome Integrity, Repair and Replication ,Crystallography, X-Ray ,DNA polymerase delta ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,Humans ,Amino Acid Sequence ,Polymerase ,Conserved Sequence ,DNA Polymerase beta ,030304 developmental biology ,0303 health sciences ,DNA clamp ,biology ,Processivity ,DNA ,Kinetics ,Biochemistry ,030220 oncology & carcinogenesis ,biology.protein ,Biophysics ,Biocatalysis ,DNA polymerase I ,DNA polymerase mu - Abstract
Differences in the substrate specificity of mammalian family X DNA polymerases are proposed to partly depend on a loop (loop 1) upstream of the polymerase active site. To examine if this is the case in DNA polymerase λ (pol λ), here we characterize a variant of the human polymerase in which nine residues of loop 1 are replaced with four residues from the equivalent position in pol β. Crystal structures of the mutant enzyme bound to gapped DNA with and without a correct dNTP reveal that the change in loop 1 does not affect the overall structure of the protein. Consistent with these structural data, the mutant enzyme has relatively normal catalytic efficiency for correct incorporation, and it efficiently participates in non-homologous end joining of double-strand DNA breaks. However, DNA junctions recovered from end-joining reactions are more diverse than normal, and the mutant enzyme is substantially less accurate than wild-type pol λ in three different biochemical assays. Comparisons of the binary and ternary complex crystal structures of mutant and wild-type pol λ suggest that loop 1 modulates pol λ’s fidelity by controlling dNTP-induced movements of the template strand and the primer-terminal 3′-OH as the enzyme transitions from an inactive to an active conformation.
- Published
- 2010
20. Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase λ in human nuclear extracts
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Thomas A. Kunkel, Luis Blanco, Lawrence F. Povirk, Katarzyna Bebenek, Miguel Garcia-Diaz, and Rui-Zhe Zhou
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Cell Extracts ,Guanine ,DNA Repair ,Base Pair Mismatch ,DNA repair ,DNA polymerase ,Base pair ,Recombinant Fusion Proteins ,DNA polymerase beta ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genetics ,Humans ,DNA Breaks, Double-Stranded ,DNA Polymerase beta ,Polymerase ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,biology ,Nucleic Acid Enzymes ,Molecular biology ,DNA polymerase lambda ,Thymine ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,DNA ,HeLa Cells - Abstract
Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3' overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase lambda. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or -GTA) 3' overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote error-prone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase lambda was replaced with that of polymerase beta, could not substitute for polymerase lambda in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.
- Published
- 2008
21. Substrate-induced DNA strand misalignment during catalytic cycling by DNA polymerase λ
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Lars C. Pedersen, Miguel Garcia-Diaz, Meredith C. Foley, Katarzyna Bebenek, Tamar Schlick, and Thomas A. Kunkel
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DNA Repair ,DNA polymerase ,DNA repair ,Scientific Report ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,D-loop ,DNA strand slippage ,Genetics ,Humans ,Computer Simulation ,Molecular Biology ,Polymerase ,DNA Polymerase beta ,030304 developmental biology ,Transcription bubble ,0303 health sciences ,Alanine ,Binding Sites ,biology ,Lysine ,Hydrogen Bonding ,DNA ,Templates, Genetic ,0104 chemical sciences ,single nucleotide deletions ,chemistry ,Amino Acid Substitution ,Models, Chemical ,Coding strand ,Biophysics ,biology.protein ,indels ,Nucleic Acid Conformation ,Primer (molecular biology) ,Gene Deletion - Abstract
The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase lambda bound to a primer-template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage.
- Published
- 2008
22. Role of the catalytic metal during polymerization by DNA polymerase lambda
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Miguel Garcia-Diaz, Katarzyna Bebenek, Thomas A. Kunkel, Joseph M. Krahn, and Lars C. Pedersen
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Models, Molecular ,Protein Conformation ,DNA polymerase ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Article ,Phosphates ,chemistry.chemical_compound ,Ribose ,Humans ,Nucleotide ,Molecular Biology ,DNA Polymerase beta ,Polymerase ,chemistry.chemical_classification ,Manganese ,Binding Sites ,biology ,Leaving group ,Active site ,DNA ,Cell Biology ,DNA polymerase lambda ,Crystallography ,chemistry ,Mutagenesis ,biology.protein ,Crystallization ,Protein Binding - Abstract
The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.
- Published
- 2007
23. RNA-templated DNA repair
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Dmitry A. Gordenin, Francesca Storici, Michael A. Resnick, Katarzyna Bebenek, and Thomas A. Kunkel
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DNA Replication ,DNA Repair ,DNA repair ,Base pair ,DNA polymerase ,Oligonucleotides ,RNA-dependent RNA polymerase ,DNA-Directed DNA Polymerase ,Saccharomyces cerevisiae ,Article ,chemistry.chemical_compound ,Transcription (biology) ,DNA Breaks, Double-Stranded ,Multidisciplinary ,biology ,fungi ,RNA ,DNA ,Templates, Genetic ,Molecular biology ,Cell biology ,chemistry ,biology.protein ,Primase - Abstract
Although RNA is used to synthesize DNA by specialized enzymes such as reverse transcriptase and telomerase, it has never been shown to be used directly in DNA repair. Here Resnick and colleagues find that short RNA oligonucleotides which are complementary to the sequences at a DNA double-strand break can serve as a template for repair by the normal replicative DNA polymerases, suggesting that information contained in an RNA molecule could be transferred into the genome. RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons1 and in the elongation of telomeres2. Despite its abundance in the nucleus, there has been no evidence for a direct role of RNA as a template in the repair of any chromosomal DNA lesions, including DNA double-strand breaks (DSBs), which are repaired in most organisms by homologous recombination or by non-homologous end joining3. An indirect role for RNA in DNA repair, following reverse transcription and formation of a complementary DNA, has been observed in the non-homologous joining of DSB ends4,5. In the yeast Saccharomyces cerevisiae, in which homologous recombination is efficient3, RNA was shown to mediate recombination, but only indirectly through a cDNA intermediate6,7 generated by the reverse transcriptase function of Ty retrotransposons in Ty particles in the cytoplasm8. Although pairing between duplex DNA and single-strand (ss)RNA can occur in vitro9,10 and in vivo11, direct homologous exchange of genetic information between RNA and DNA molecules has not been observed. We show here that RNA can serve as a template for DNA synthesis during repair of a chromosomal DSB in yeast. The repair was accomplished with RNA oligonucleotides complementary to the broken ends. This and the observation that even yeast replicative DNA polymerases such as α and δ can copy short RNA template tracts in vitro demonstrate that RNA can transfer genetic information in vivo through direct homologous interaction with chromosomal DNA.
- Published
- 2007
24. Multiple Functions of DNA Polymerases
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Miguel Garcia-Diaz and Katarzyna Bebenek
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Genetics ,biology ,DNA polymerase ,DNA repair ,DNA replication ,Eukaryotic DNA replication ,Plant Science ,DNA polymerase delta ,Article ,chemistry.chemical_compound ,chemistry ,biology.protein ,DNA supercoil ,Polymerase ,DNA - Abstract
The primary role of DNA polymerases is to accurately and efficiently replicate the genome in order to ensure the maintenance of the genetic information and its faithful transmission through generations. This is not a simple task considering the size of the genome and its constant exposure to endogenous and environmental DNA damaging agents. Thus, a number of DNA repair pathways operate in cells to protect the integrity of the genome. In addition to their role in replication, DNA polymerases play a central role in most of these pathways. Given the multitude and the complexity of DNA transactions that depend on DNA polymerase activity, it is not surprising that cells in all organisms contain multiple highly specialized DNA polymerases, the majority of which have only recently been discovered. Five DNA polymerases are now recognized in Escherichia coli, 8 in Saccharomyces cerevisiae, and at least 15 in humans. While polymerases in bacteria, yeast and mammalian cells have been extensively studied much less is known about their counterparts in plants. For example, the plant model organism Arabidopsis thaliana is thought to contain 12 DNA polymerases, whose functions are mostly unknown. Here we review the properties and functions of DNA polymerases focusing on yeast and mammalian cells but paying special attention to the plant enzymes and the special circumstances of replication and repair in plant cells.
- Published
- 2007
25. Creative template-dependent synthesis by human polymerase mu
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Thomas A. Kunkel, Lars C. Pedersen, Rajendrakumar A. Gosavi, Andrea F. Moon, and Katarzyna Bebenek
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DNA Repair ,Stereochemistry ,DNA polymerase ,DNA polymerase II ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,Catalysis ,Protein Structure, Secondary ,Substrate Specificity ,Humans ,DNA Polymerase beta ,Multidisciplinary ,biology ,Nucleotides ,DNA replication ,Base excision repair ,Processivity ,DNA ,Sequence Analysis, DNA ,DNA polymerase lambda ,Kinetics ,Biochemistry ,PNAS Plus ,biology.protein ,Nucleic Acid Conformation ,DNA polymerase mu ,DNA Damage - Abstract
Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol µ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol µ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol µ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol µ “skips” the first available template nucleotide, instead using the template base at the 5′ end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3′-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.
- Published
- 2015
26. Alternative solutions and new scenarios for translesion DNA synthesis by human PrimPol
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Katarzyna Bebenek, Patricia A. Calvo, María I. Martínez-Jiménez, Guillermo Sastre-Moreno, Sara García-Gómez, Luis Blanco, Alberto Díaz-Talavera, Thomas A. Kunkel, Fundación Ramón Areces, National Institute of Environmental Health Sciences (US), Ministerio de Economía y Competitividad (España), and Comunidad de Madrid
- Subjects
DNA Replication ,DNA Repair ,DNA polymerase ,DNA damage ,Translesion synthesisa ,8oxoG ,DNA-Directed DNA Polymerase ,DNA Primase ,Biochemistry ,Transcription (biology) ,Cations ,Humans ,Nucleotide ,Molecular Biology ,Polymerase ,Genetics ,chemistry.chemical_classification ,Manganese ,biology ,DNA synthesis ,DNA ,Cell Biology ,Multifunctional Enzymes ,Lesion bypass ,chemistry ,biology.protein ,Nucleic Acid Conformation ,PrimPol ,Primase ,Primer (molecular biology) ,DNA Damage - Abstract
© 2015 Elsevier B.V. PrimPol is a recently described DNA polymerase that has the virtue of initiating DNA synthesis. In addition of being a sensu stricto DNA primase, PrimPol's polymerase activity has a large capacity to tolerate different kind of lesions. The different strategies used by PrimPol for DNA damage tolerance are based on its capacity to >read> certain lesions, to skip unreadable lesions, and as an ultimate solution, to restart DNA synthesis beyond the lesion thus acting as a TLS primase. This lesion bypass potential, revised in this article, is strengthened by the preferential use of moderate concentrations of manganese ions as the preferred metal activator. We show here that PrimPol is able to extend RNA primers with ribonucleotides, even when bypassing 8oxoG lesions, suggesting a potential new scenario for PrimPol as a TLS polymerase assisting transcription. We also show that PrimPol displays a high degree of versatility to accept or induce distortions of both primer and template strands, creating alternative alignments based on microhomology that would serve to skip unreadable lesions and to connect separate strands. In good agreement, PrimPol is highly prone to generate indels at short nucleotide repeats. Finally, an evolutionary view of the relationship between translesion synthesis and primase functions is briefly discussed., This work wassupported by Comunidad de Madrid (S2010/BMD-2361), Spanish Ministry of Economy and Competitiveness (BFU2012-37969) toL.B., by the Division of Intramural Research of the US NationalInstitutes of Health (NIH), National Institute of Environmental Health Sciences project Z01 ES065070 to T.A.K., and by an Institu-tional grant to Centro de Biología Molecular ‘Severo Ochoa’ from Fundación Ramón Areces.
- Published
- 2015
27. Structural insight into the substrate specificity of DNA Polymerase μ
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Xuejun Zhong, Dale A. Ramsden, Miguel Garcia-Diaz, Bryan J Davis, Andrea F. Moon, Katarzyna Bebenek, Thomas A. Kunkel, and Lars C. Pedersen
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Models, Molecular ,DNA Repair ,DNA polymerase ,Stereochemistry ,DNA polymerase II ,Molecular Sequence Data ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,Substrate Specificity ,Mice ,Structural Biology ,Animals ,Thymine Nucleotides ,Amino Acid Sequence ,Molecular Biology ,Polymerase ,Binding Sites ,DNA clamp ,biology ,DNA replication ,DNA ,Processivity ,Molecular biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Amino Acid Substitution ,biology.protein ,Sequence Alignment ,DNA polymerase mu ,Dideoxynucleotides - Abstract
DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.
- Published
- 2006
28. Structural Analysis of Strand Misalignment during DNA Synthesis by a Human DNA Polymerase
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Miguel Garcia-Diaz, Katarzyna Bebenek, Thomas A. Kunkel, Lars C. Pedersen, and Joseph M. Krahn
- Subjects
Models, Molecular ,Genetics ,Binding Sites ,DNA clamp ,DNA Repair ,biology ,DNA polymerase ,DNA repair ,Base pair ,Biochemistry, Genetics and Molecular Biology(all) ,DNA polymerase II ,DNA ,DNA polymerase delta ,Catalysis ,General Biochemistry, Genetics and Molecular Biology ,Protein Structure, Tertiary ,Kinetics ,biology.protein ,Humans ,DNA polymerase mu ,DNA Polymerase beta ,Gene Deletion ,Polymerase - Abstract
Insertions and deletions in coding sequences can alter the reading frame of genes and have profound biological consequences. In 1966, Streisinger proposed that these mutations result from strand slippage, which in repetitive sequences generates misaligned intermediates stabilized by correct base pairing that support polymerization. We report here crystal structures of human DNA polymerase lambda, which frequently generates deletion mutations, bound to such intermediates. Each contains an extrahelical template nucleotide upstream of the active site. Surprisingly, the extra nucleotide, even when combined with an adjacent mismatch, does not perturb polymerase active site geometry, which is indistinguishable from that for correctly aligned strands. These structures reveal how pol lambda can polymerize on substrates with minimal homology during repair of double-strand breaks and represent strand-slippage intermediates consistent with Streisinger's classical hypothesis. They are thus relevant to the origin of single-base deletions, a class of mutations that can confer strong biological phenotypes.
- Published
- 2006
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29. A Gradient of Template Dependence Defines Distinct Biological Roles for Family X Polymerases in Nonhomologous End Joining
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Jody M. Havener, Stephanie A. Nick McElhinny, Dale A. Ramsden, Katarzyna Bebenek, Thomas A. Kunkel, Miguel Garcia-Diaz, Raquel Juárez, Barbara L. Kee, and Luis Blanco
- Subjects
Models, Molecular ,DNA Repair ,viruses ,Molecular Sequence Data ,Gene Expression ,Context (language use) ,DNA-Directed DNA Polymerase ,Transfection ,Cell Line ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Immunoglobulin kappa-Chains ,Mice ,0302 clinical medicine ,DNA Nucleotidylexotransferase ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Polymerase ,DNA Polymerase beta ,030304 developmental biology ,Genetics ,Gene Rearrangement ,Recombination, Genetic ,0303 health sciences ,B-Lymphocytes ,biology ,Sequence Homology, Amino Acid ,fungi ,DNA ,Templates, Genetic ,Cell Biology ,DNA polymerase lambda ,Recombinant Proteins ,Cell biology ,Non-homologous end joining ,enzymes and coenzymes (carbohydrates) ,chemistry ,030220 oncology & carcinogenesis ,Coding strand ,embryonic structures ,biology.protein ,Immunoglobulin Joining Region ,Primer (molecular biology) ,Recombination - Abstract
Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.
- Published
- 2005
- Full Text
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30. Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV
- Author
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Miguel Garcia-Diaz, Thomas A. Kunkel, Katarzyna Bebenek, and Steven R. Patishall
- Subjects
Saccharomyces cerevisiae Proteins ,DNA Repair ,DNA polymerase ,DNA repair ,viruses ,Molecular Sequence Data ,DNA-Directed DNA Polymerase ,Saccharomyces cerevisiae ,Biochemistry ,DNA polymerase delta ,Substrate Specificity ,Amino Acid Sequence ,DNA, Fungal ,Molecular Biology ,DNA Polymerase beta ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,DNA replication ,Cell Biology ,Base excision repair ,Processivity ,Ribonucleotides ,Molecular biology ,Recombinant Proteins ,Kinetics ,Lac Operon ,Mutation ,DNA polymerase IV ,biology.protein ,Phosphorus-Oxygen Lyases ,DNA polymerase mu - Abstract
Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol beta and pol lambda, but not pol mu, pol IV has intrinsic 5'-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol beta and pol lambda, the gap-filling activity of pol IV is not enhanced by a 5'-phosphate on the downstream primer but is stimulated by a 5'-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol mu but not pol beta or pol lambda. Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.
- Published
- 2005
31. Pol ι Is a Candidate for the Mouse Pulmonary Adenoma Resistance 2 Locus, a Major Modifier of Chemically Induced Lung Neoplasia
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Theodora R. Devereux, Scott D. McCulloch, Ming You, Min Wang, Thomas A. Kunkel, Colleen H. Anna, Yian Wang, Haris G. Vikis, Wanda Holliday, Kun-Liang Guan, and Katarzyna Bebenek
- Subjects
Adenoma ,Cancer Research ,Candidate gene ,Lung Neoplasms ,Mice, Inbred A ,DNA polymerase ,Molecular Sequence Data ,Single-nucleotide polymorphism ,DNA-Directed DNA Polymerase ,medicine.disease_cause ,Polymorphism, Single Nucleotide ,Proto-Oncogene Proteins p21(ras) ,Mice ,Proto-Oncogene Proteins ,Gene expression ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Lung ,Gene ,Regulation of gene expression ,Mice, Inbred BALB C ,Sequence Homology, Amino Acid ,biology ,Molecular biology ,Immunity, Innate ,Gene Expression Regulation, Neoplastic ,Alternative Splicing ,Oncology ,DNA Polymerase iota ,ras Proteins ,biology.protein ,Carcinogenesis ,DNA Polymerase Iota - Abstract
In this study, we performed systematic candidate gene analyses of the Pulmonary adenoma resistance 2 locus. Differential gene expression in lung tissues and nucleotide polymorphisms in coding regions between A/J and BALB/cJ mice were examined using reverse transcription-PCR and direct sequencing. Although not all genes in the interval were analyzed at this moment due to the recent database updating, we have found that the Pol ι gene, encoding the DNA polymerase ι, contains 25 nucleotide polymorphisms in its coding region between A/J and BALB/cJ mice, resulting in a total of ten amino acid changes. Primer extension assays with purified BALB/cJ and A/J proteins in vitro demonstrate that both forms of Pol ι are active but that they may differ in substrate discrimination, which may affect the formation of Kras2 mutations in mouse lung tumors. Altered expression of POL ι protein and an amino acid-changing nucleotide polymorphism were observed in human lung cancer cells, suggesting a possible role in the development of lung cancer. Thus, our data support the Pol ι gene as a modifier of lung tumorigenesis by altering DNA polymerase activity.
- Published
- 2004
32. Implication of DNA Polymerase λ in Alignment-based Gap Filling for Nonhomologous DNA End Joining in Human Nuclear Extracts
- Author
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Jae Wan Lee, Lawrence F. Povirk, Miguel Garcia-Diaz, Zhigang Wang, Katarzyna Bebenek, Thomas A. Kunkel, Luis Blanco, and Tong Zhou
- Subjects
Cell Nucleus ,DNA clamp ,Base Sequence ,biology ,DNA polymerase ,DNA polymerase II ,DNA ,Cell Biology ,Biochemistry ,Molecular biology ,DNA polymerase delta ,DNA polymerase lambda ,Substrate Specificity ,DNA-Binding Proteins ,biology.protein ,Humans ,DNA polymerase I ,Molecular Biology ,DNA polymerase mu ,DNA Polymerase beta ,Polymerase ,HeLa Cells - Abstract
Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.
- Published
- 2004
33. The Frameshift Infidelity of Human DNA Polymerase λ
- Author
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Miguel Garcia-Diaz, Thomas A. Kunkel, Katarzyna Bebenek, and Luis Blanco
- Subjects
Genetics ,biology ,DNA polymerase ,viruses ,DNA polymerase II ,DNA replication ,Cell Biology ,Base excision repair ,Processivity ,Biochemistry ,DNA polymerase delta ,Molecular biology ,DNA polymerase lambda ,biology.protein ,Molecular Biology ,DNA polymerase mu - Abstract
DNA polymerase λ (Pol λ) is a member of the Pol X family having properties in common with several other mammalian DNA polymerases. To obtain clues to possible functions in vivo, we have determined the fidelity of DNA synthesis by human Pol λ. The results indicate that the average single-base deletion error rate of Pol λ is higher than those of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol λ generates single-base deletions at average rates that substantially exceed base substitution rates. Moreover, the sequence specificity for single-base deletions made by Pol λ is different from that of other DNA polymerases and reveals that Pol λ readily uses template-primers with limited base pair homology at the primer terminus. This ability, together with an ability to fill short gaps in DNA at low dNTP concentrations, is consistent with a role for mammalian Pol λ in non-homologous end-joining. This may include non-homologous end-joining of strand breaks resulting from DNA damage, because Pol λ has intrinsic 5′,2′-deoxyribose-5-phosphate lyase activity.
- Published
- 2003
34. Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase ι by Controlled Proteolysis
- Author
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Katarzyna Bebenek, Thomas A. Kunkel, David D. Shock, Roger Woodgate, Esther W. Hou, Samuel H. Wilson, William A. Beard, and Rajendra Prasad
- Subjects
Time Factors ,DNA Repair ,DNA polymerase ,DNA polymerase II ,DNA-Directed DNA Polymerase ,Biochemistry ,DNA polymerase delta ,Chymotrypsin ,Humans ,Lyase activity ,Molecular Biology ,DNA Polymerase beta ,Polymerase ,Glutathione Transferase ,Binding Sites ,biology ,Cell Biology ,Processivity ,Base excision repair ,Lyase ,Molecular biology ,Protein Structure, Tertiary ,Kinetics ,Cross-Linking Reagents ,DNA Polymerase iota ,biology.protein ,Phosphorus-Oxygen Lyases ,Protein Binding ,Subcellular Fractions - Abstract
Human DNA polymerase iota (pol iota) is a member of the Y-family of low fidelity lesion bypass DNA polymerases. In addition to a probable role in DNA lesion bypass, this enzyme has recently been shown to be required for somatic hypermutation in human B-cells. We found earlier that human pol iota has deoxyribose phosphate (dRP) lyase activity and unusual specificity for activity during DNA synthesis, suggesting involvement in specialized forms of base excision repair (BER). Here, mapping of the domain structure of human pol iota by controlled proteolysis revealed that the enzyme has a 48-kDa NH2-terminal domain and a protease resistant 40-kDa "core domain" spanning residues Met79 to approximately Met445. A covalently cross-linked pol iota-DNA complex, representing a trapped intermediate in the dRP lyase reaction, was subjected to controlled proteolysis. Cross-linking was mapped to the 40-kDa core domain, indicating that the dRP lyase active site is in this region. To further evaluate the BER capacity of the enzyme, the dRP lyase and DNA polymerase activities were characterized on DNA substrates representing BER intermediates, and we found that pol iota was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase beta null cell extract.
- Published
- 2003
35. 32nd Annual Meeting of European Environmental Mutagen Society
- Author
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Andrew Collins, Serge Boiteux, Jacques Laval, Zygmunt Ciesla, Barbara Tudek, Helmut Bartsch, Hideo Shinagawa, Katarzyna Bebenek, Leon F.H. Mullenders, Krzysztof Szyfter, Marcin Kruszewski, Celina Janion, and Albert A. van Zeeland
- Subjects
Genetics ,0303 health sciences ,DNA damage ,Mutagen ,Cell Biology ,Biology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Environmental protection ,030220 oncology & carcinogenesis ,medicine ,Molecular Biology ,030304 developmental biology - Published
- 2003
36. Low Fidelity DNA Synthesis by a Y Family DNA Polymerase Due to Misalignment in the Active Site
- Author
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Thomas A. Kunkel, Robert J. Kokoska, Katarzyna Bebenek, François Boudsocq, and Roger Woodgate
- Subjects
Guanine ,DNA polymerase ,Base pair ,DNA polymerase II ,Molecular Sequence Data ,Biochemistry ,Sulfolobus ,Cytosine ,Structure-Activity Relationship ,Sequence Homology, Nucleic Acid ,Cysteine ,Molecular Biology ,DNA Polymerase beta ,Genetics ,Binding Sites ,DNA clamp ,Base Sequence ,biology ,DNA replication ,Cell Biology ,Kinetics ,Mutation ,DNA polymerase IV ,biology.protein ,Primase ,DNA polymerase mu ,Gene Deletion - Abstract
Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a member of the Y family of DNA polymerases whose crystal structure has recently been solved. As a model for other evolutionarily conserved Y family members that perform translesion DNA synthesis and have low fidelity, we describe here the base substitution and frameshift fidelity of DNA synthesis by Dpo4. Dpo4 generates all 12 base-base mismatches at high rates, 11 of which are similar to those of its human homolog, DNA polymerase kappa. This result is consistent with the Dpo4 structure, implying lower geometric selection for correct base pairs. Surprisingly, Dpo4 generates C.dCMP mismatches at an unusually high average rate and preferentially at cytosine flanked by 5'-template guanine. Dpo4 also has very low frameshift fidelity and frequently generates deletions of even noniterated nucleotides, especially cytosine flanked by a 5'-template guanine. Both unusual features of error specificity suggest that Dpo4 can incorporate dNTP precursors when two template nucleotides are present in the active site binding pocket. These results have implications for mutagenesis resulting from DNA synthesis by Y family polymerases.
- Published
- 2002
37. Visions & Reflections¶Family growth: the eukaryotic DNA polymerase revolution
- Author
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Katarzyna Bebenek and Thomas A. Kunkel
- Subjects
Pharmacology ,Genetics ,DNA clamp ,biology ,Chemistry ,DNA polymerase ,DNA polymerase II ,Eukaryotic DNA replication ,Cell Biology ,DNA polymerase delta ,Molecular biology ,Cellular and Molecular Neuroscience ,biology.protein ,Molecular Medicine ,DNA polymerase I ,Molecular Biology ,DNA polymerase mu ,Polymerase - Published
- 2002
38. Structures of the Leishmania infantum polymerase beta
- Author
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Edison Mejia, Thomas A. Kunkel, Matthew J. Burak, Katarzyna Bebenek, Miguel Garcia-Diaz, Ana Alonso, Vicente Larraga, National Institutes of Health (US), Fundación Ramón Areces, Ministerio de Ciencia, Innovación y Universidades (España), Mejía Edison, Alonso, Ana, Larraga, Vicente, Kunkel, Thomas A., Bebenek, Katarzyna, García-Díaz, Miguel, Mejía Edison [0000-0002-6565-3737], Alonso, Ana [0000-0002-1228-7331], Larraga, Vicente [0000-0003-1260-7400], Kunkel, Thomas A. [0000-0002-9900-1788], Bebenek, Katarzyna [0000-0002-3263-7992], and García-Díaz, Miguel [0000-0003-1605-6861]
- Subjects
Models, Molecular ,Protein Folding ,DNA polymerase ,Protein Conformation ,DNA polymerase II ,DNA repair ,Crystallography, X-Ray ,Biochemistry ,DNA polymerase delta ,Protein Structure, Secondary ,Article ,Catalytic Domain ,Leishmania infantum ,Protein Structure, Quaternary ,Molecular Biology ,Leishmaniasis ,Polymerase ,DNA Polymerase beta ,Genetics ,DNA clamp ,biology ,Sequence Homology, Amino Acid ,DNA replication ,Cell Biology ,Processivity ,Molecular biology ,Mutation ,biology.protein ,DNA polymerase mu ,Family X - Abstract
9 p.-4 fig.-4 tab., Protozoans of the genus Leishmania, the pathogenic agent causing leishmaniasis, encode the family X DNA polymerase Li Pol β. Here, we report the first crystal structures of Li Pol β. Our pre- and post-catalytic structures show that the polymerase adopts the common family X DNA polymerase fold. However, in contrast to other family X DNA polymerases, the dNTP-induced conformational changes in Li Pol β are much more subtle. Moreover, pre- and post-catalytic structures reveal that Li Pol β interacts with the template strand through a nonconserved, variable region known as loop3. Li Pol β Δloop3 mutants display a higher catalytic rate, catalytic efficiency and overall error rates with respect to WT Li Pol β. These results further demonstrate the subtle structural variability that exists within this family of enzymes and provides insight into how this variability underlies the substantial functional differences among their members., This work was supported by R01-GM100021 and R00 ES015421 to MGD. AA and VL thank the Fundacion Ramon Areces and MICINN (AGL2010-21806-C02-01) for funding.
- Published
- 2014
39. Sustained active site rigidity during synthesis by human DNA polymerase μ
- Author
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Lars C. Pedersen, Thomas A. Kunkel, Katarzyna Bebenek, Andrea F. Moon, Dale A. Ramsden, and John M. Pryor
- Subjects
Models, Molecular ,DNA Repair ,DNA polymerase ,DNA polymerase II ,Electrons ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,Catalysis ,Article ,Substrate Specificity ,Structural Biology ,Catalytic Domain ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,Polymerase ,DNA clamp ,biology ,Nucleotides ,DNA replication ,Processivity ,Kinetics ,enzymes and coenzymes (carbohydrates) ,Biochemistry ,Mutation ,biology.protein ,Biophysics ,Crystallization ,DNA polymerase mu ,Protein Binding - Abstract
DNA polymerase μ (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3' ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol μ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible loop 1 region repositions upon DNA binding. Pol μ variants with changes in loop 1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific loop 1 residues contribute to Pol μ's unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3' ends.
- Published
- 2014
- Full Text
- View/download PDF
40. Identification of an Intrinsic 5′-Deoxyribose-5-phosphate Lyase Activity in Human DNA Polymerase λ
- Author
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Katarzyna Bebenek, Miguel Garcia-Diaz, Luis Blanco, and Thomas A. Kunkel
- Subjects
biology ,DNA polymerase ,Cell Biology ,Base excision repair ,Processivity ,Biochemistry ,DNA polymerase delta ,Molecular biology ,DNA polymerase lambda ,5'-deoxyribose-5-phosphate lyase activity ,biology.protein ,Molecular Biology ,DNA polymerase mu ,Nucleotide excision repair - Abstract
Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5′-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase β (Pol β). DNA polymerase λ (Pol λ) is a recently identified eukaryotic DNA polymerase that is homologous to Pol β. We show here that human Pol λ exhibits dRP lyase, but not AP lyase, activityin vitro and that this activity is consistent with a β-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys310 of Pol λ is the main nucleophile involved in the reaction. The dRP lyase activity of Pol λ, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitroreconstituted BER reaction. These results suggest that Pol λ may participate in “single-nucleotide” base excision repair in mammalian cells.
- Published
- 2001
41. Error rate and specificity of human and murine DNA polymerase η
- Author
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Chikahide Masutani, Toshiro Matsuda, Thomas A. Kunkel, Igor B. Rogozin, Katarzyna Bebenek, and Fumio Hanaoka
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DNA Replication ,Exonuclease ,Base Pair Mismatch ,DNA polymerase ,DNA Mutational Analysis ,Molecular Sequence Data ,Somatic hypermutation ,DNA-Directed DNA Polymerase ,DNA polymerase eta ,Substrate Specificity ,Mice ,chemistry.chemical_compound ,Structural Biology ,Animals ,Humans ,Point Mutation ,Frameshift Mutation ,Molecular Biology ,Sequence Deletion ,Genetics ,Base Sequence ,Genes, Immunoglobulin ,DNA synthesis ,biology ,Templates, Genetic ,Processivity ,Kinetics ,Lac Operon ,chemistry ,Mutagenesis ,biology.protein ,Proofreading ,DNA ,DNA Damage - Abstract
We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.
- Published
- 2001
42. The Bloom's Syndrome Protein (BLM) Interacts with MLH1 but Is Not Required for DNA Mismatch Repair
- Author
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Thomas A. Kunkel, Kathleen H. Goss, Gregory Langland, Joanna Groden, Katarzyna Bebenek, Jennifer J. Kordich, Kate Lillard-Wetherell, and Jenette Creaney
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DNA Replication ,Male ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA Repair ,Base Pair Mismatch ,DNA repair ,RecQ helicase ,Blotting, Western ,Biochemistry ,Cell Line ,Two-Hybrid System Techniques ,Tumor Cells, Cultured ,Humans ,Child ,Molecular Biology ,Gene ,Cells, Cultured ,Adaptor Proteins, Signal Transducing ,Adenosine Triphosphatases ,Cell Nucleus ,Recombination, Genetic ,Genetics ,RecQ Helicases ,biology ,urogenital system ,DNA Helicases ,DNA replication ,Nuclear Proteins ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,Precipitin Tests ,Neoplasm Proteins ,Mutation ,biology.protein ,DNA mismatch repair ,Carrier Proteins ,K562 Cells ,MutL Protein Homolog 1 ,HeLa Cells ,Protein Binding - Abstract
Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.
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- 2001
43. A Molecular Dynamics Model of HIV-1 Reverse Transcriptase Complexed with DNA: Comparison with Experimental Structures
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Thomas A. Kunkel, Katarzyna Bebenek, Lee G. Pedersen, Samuel H. Wilson, Thomas A. Darden, Leping Li, and William A. Beard
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Chemistry ,Organic Chemistry ,Crystal structure ,Catalysis ,Reverse transcriptase ,Force field (chemistry) ,Computer Science Applications ,Inorganic Chemistry ,Root mean square ,Molecular dynamics ,chemistry.chemical_compound ,Crystallography ,Computational Theory and Mathematics ,Physical and Theoretical Chemistry ,Ternary complex ,Protein secondary structure ,DNA - Abstract
We have built a molecular dynamics model for human immunodeficiency virus (HIV-1) reverse transcriptase (RT) complexed with a 19/18-mer template/primer by combining the structural information of a low resolution crystal structure of a HIV-1 RT/DNA complex (1hmi) with that of a high resolution crystal structure of unliganded HIV-1 RT (1rtj). The process involved slow forcing of the α-carbons of 1rtj onto those of 1hmi using constrained MD simulations, while immersing the protein in aqueous solution. A similar technique was used to build the bent all-atom DNA duplex, which was then docked into the modeled protein. The resulting model complex was refined using molecular dynamics simulation with the Particle-mesh Ewald method employed to accommodate long-range electrostatic interactions. New parameters of the Amber force field that affect DNA twist are tested and largely validated. The model has been used successfully to explain the results of vertical scanning mutagenesis of residue 266 (Trp266). Recently, the low resolution crystal structure of the HIV-1 RT/DNA complex has been refined to a 2.8 A resolution (2hmi) and a crystal structure of a HIV-1/RT/dTTP ternary complex has been determined at 3.2 A resolution (1rtd). A detailed structural comparison of the prior model structure and the two experimental structures becomes possible. Overall, the three structures share many similarities. The root mean square deviations of the α-carbons for the individual subdomains among the three structures are within the same ranges. The secondary structure assignments in the three structures are nearly identical. Key protein-DNA contacts such as those in the region of the primer grip are also similar in the three structures.
- Published
- 2000
44. The base substitution fidelity of HIV-1 reverse transcriptase on DNA and RNA templates probed with 8-oxo-deoxyguanosine triphosphate
- Author
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Jayne C. Boyer, Katarzyna Bebenek, and Thomas A. Kunkel
- Subjects
Models, Molecular ,Base Pair Mismatch ,Health, Toxicology and Mutagenesis ,Molecular Sequence Data ,chemistry.chemical_compound ,Genetics ,Humans ,Transversion ,Molecular Biology ,Polymerase ,Binding Sites ,Base Sequence ,biology ,RNA-Directed DNA Polymerase ,Point mutation ,Nucleic Acid Heteroduplexes ,Nucleic acid sequence ,Deoxyguanine Nucleotides ,RNA ,DNA ,Templates, Genetic ,Molecular biology ,HIV Reverse Transcriptase ,Recombinant Proteins ,Reverse transcriptase ,chemistry ,Biochemistry ,Mutation ,HIV-1 ,biology.protein - Abstract
We have used 8-O-dGTP, a mutagenic nucleotide generated by oxidative metabolism, to probe the misincorporation potential of HIV-1 reverse transcriptase (RT) during DNA synthesis templated by the same nucleotide sequence as either RNA or DNA. With either template, 8-O-dGMP was misincorporated opposite template A, yielding characteristic A--C transversions. The error rate with DNA was similar to that with RNA, suggesting that base misincorporation by the RT during first-strand and second-strand replication may contribute equally to the HIV-1 base substitution mutation rate. The rate of 8-O-dGMP misincorporation differed by more than 10-fold among the 20 adenines in the M13mp2 template where A--C transversions can be detected. The transversion distribution was similar with the two templates, indicating that the effects of flanking nucleotides on misincorporation rates were similar. This is consistent with structural and biochemical data suggesting that HIV-1 RT binds RNA x DNA and DNA x DNA template-primers in the same orientation. The similarities in error rates and distribution further indicate that, despite differences in the structures of free RNA x DNA and DNA x DNA duplexes (e.g., minor groove dimensions), the polymerase active site that assembles upon substrate binding establishes a similar degree of nucleotide selectivity with both types of template-primers. Comparison of the RT error distribution to that observed with two Pol I family DNA polymerases and a Pol alpha family polymerase revealed common hot and cold spots for misincorporation. This suggests that the local nucleotide sequence influences the nucleotide selectivity of four polymerases in a similar manner, despite their differences in structure, biochemical properties, and functions.
- Published
- 1999
45. Residues in the αH and αI Helices of the HIV-1 Reverse Transcriptase Thumb Subdomain Required for the Specificity of RNase H-catalyzed Removal of the Polypurine Tract Primer
- Author
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Judith G. Levin, Stuart F. J. Le Grice, William A. Beard, Kathryn J. Howard, Samuel H. Wilson, Thomas A. Darden, Michael D. Powell, Thomas A. Kunkel, and Katarzyna Bebenek
- Subjects
Models, Molecular ,Molecular Sequence Data ,Ribonuclease H ,Mutant ,Cleavage (embryo) ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,Retrovirus ,Protein structure ,Humans ,heterocyclic compounds ,Structural motif ,RNase H ,Molecular Biology ,Alanine ,Base Sequence ,biology ,Tryptophan ,DNA ,Cell Biology ,biology.organism_classification ,Molecular biology ,HIV Reverse Transcriptase ,Reverse transcriptase ,Kinetics ,Mutation ,HIV-1 ,biology.protein ,Nucleic Acid Conformation ,RNA ,Primer (molecular biology) - Abstract
During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.
- Published
- 1999
46. Vertical-scanning Mutagenesis of a Critical Tryptophan in the Minor Groove Binding Track of HIV-1 Reverse Transcriptase
- Author
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Thomas A. Kunkel, Katarzyna Bebenek, Samuel H. Wilson, R. Stephen Lloyd, Gary J. Latham, Rajendra Prasad, William A. Beard, and Eva Forgacs
- Subjects
biology ,DNA polymerase ,Base pair ,Mutagenesis ,Cell Biology ,Processivity ,Molecular biology ,DNA-binding protein ,Biochemistry ,Reverse transcriptase ,DNA binding site ,chemistry.chemical_compound ,chemistry ,biology.protein ,Biophysics ,Primer (molecular biology) ,Groove (joinery) ,Molecular Biology ,DNA ,Polymerase - Abstract
While sequence-specific DNA-binding proteins interact predominantly in the DNA major groove, DNA polymerases bind DNA through interactions in the minor groove that are sequence nonspecific. Through functional analyses of alanine-substituted mutant enzymes that were guided by molecular dynamics modeling of the human immunodeficiency virus type 1-reverse transcriptase and DNA complex, we previously identified a structural element in reverse transcriptase, the minor groove binding track (MGBT). The MGBT is comprised of five residues (Ile94, Gln258, Gly262, Trp266, and Gln269) which interact 2–6 base pairs upstream from the polymerase active site in the DNA minor groove and are important in DNA binding, processivity, and frameshift fidelity. These residues do not contribute equally; functional analysis of alanine mutants suggests that Trp266contributes the most to binding. To define the molecular interactions between Trp266 and the DNA minor groove, we have analyzed the properties of eight mutants, each with an alternate side chain at this position. A refined molecular dynamics model was used to calculate relative binding free energies based on apolar surface area buried upon complex formation. In general, there was a strong correlation between the relative calculated binding free energies for the alternate residue 266 side chains and the magnitude of the change in the properties which reflect template-primer interactions (template-primer dissociation rate constant, K i,AZTTP, processivity, and frameshift fidelity). This correlation suggests that hydrophobic interactions make a major contribution to the stability of the polymerase-DNA complex. Additionally, tyrosine and arginine substitutions resulted in mutant enzymes with DNA binding properties better than predicted by buried surface area alone, suggesting that hydrogen bonding could also play a role in DNA binding at this position.
- Published
- 1998
47. Highly Mutagenic Bypass Synthesis by T7 RNA Polymerase of Site-specific Benzo[a]pyrene Diol Epoxide-adducted Template DNA
- Author
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Constance M. Harris, Katarzyna Bebenek, Samuel E. Bennett, Kathryn Remington, and Thomas M. Harris
- Subjects
Transcription, Genetic ,Guanine ,7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide ,Diol ,Reversion ,Biochemistry ,DNA Adducts ,Viral Proteins ,chemistry.chemical_compound ,Isomerism ,Bacteriophage T7 ,medicine ,T7 RNA polymerase ,Nucleotide ,Molecular Biology ,chemistry.chemical_classification ,Base Sequence ,DNA-Directed RNA Polymerases ,Templates, Genetic ,Cell Biology ,Molecular biology ,chemistry ,Benzo(a)pyrene ,Pyrene ,DNA ,Mutagens ,medicine.drug - Abstract
We have previously developed an in vitro system that allows quantitative evaluation of the fidelity of transcription during synthesis on a natural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N6-position with (-)-anti-trans- or (+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full-length run-off transcripts. The extent of bypass was found to be 32% for the (-)-anti-trans-isomer and 18% for the (+)-anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synthesis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell.
- Published
- 1998
48. Probing Structure/Function Relationships of HIV-1 Reverse Transcriptase with Styrene Oxide N2-Guanine Adducts
- Author
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Thomas A. Kunkel, William A. Beard, Katarzyna Bebenek, Gary J. Latham, Eva Forgacs, R. Stephen Lloyd, Samuel H. Wilson, and Rajendra Prasad
- Subjects
DNA Replication ,Models, Molecular ,Guanine ,Stereochemistry ,DNA polymerase ,Molecular Sequence Data ,Mutant ,Biochemistry ,DNA Adducts ,chemistry.chemical_compound ,Isomerism ,Styrene oxide ,Molecular Biology ,Polymerase ,Base Sequence ,biology ,Oligonucleotide ,Chemistry ,DNA ,Templates, Genetic ,Cell Biology ,HIV Reverse Transcriptase ,Reverse transcriptase ,biology.protein ,Epoxy Compounds ,Nucleic Acid Conformation - Abstract
Details of the interactions between the human immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA were probed both by introducing site-specific and stereospecific modifications into DNA and by altering the structure of potential critical residues in the polymerase. Unadducted 11-mer DNAs and 11-mer DNAs containing R and S enantiomers of styrene oxide at N2-guanine were ligated with two additional oligonucleotides to create 63-mers that served as templates for HIV-1 reverse transcriptase replication. Oligonucleotides that primed synthesis 5 bases 3' to the adducts could be extended up to 1 base 3' and opposite the lesion. However, when the positions of the 3'-OH of the priming oligonucleotides were placed 1, 2, 3, 4, 5, and 6 bases downstream of the styrene oxide guanine adducts, replication was initiated, only to be blocked after incorporating 4, 5, 6, and 7 bases beyond the lesion. The sites of this adduct-induced termination corresponded to the position of the DNA where alpha-helix H makes contact with the DNA minor groove, 3-5 bases upstream of the growing 3' end. In addition, mutants of the polymerase in alpha-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that alpha-helix H is a sensitive monitor of modifications in the minor groove of newly synthesized template-primer DNA several bases distal to the 3'-OH.
- Published
- 1997
49. A minor groove binding track in reverse transcriptase
- Author
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Bruce A. Luxon, David G. Gorenstein, Leping Li, Thomas A. Darden, Samuel H. Wilson, Katarzyna Bebenek, William A. Beard, Rajendra Prasad, and Thomas A. Kunkel
- Subjects
Structural Biology ,Chemistry ,Track (disk drive) ,Genetics ,Biophysics ,Biochemistry ,Virology ,Reverse transcriptase ,Minor groove - Abstract
Evidence is presented indicating that processive synthesis by HIV-1 reverse transcriptase involves interactions between the minor groove of the template-primer and a discrete protein structural element, the minor groove binding track (MGBT).
- Published
- 1997
50. Reduced Frameshift Fidelity and Processivity of HIV-1 Reverse Transcriptase Mutants Containing Alanine Substitutions in Helix H of the Thumb Subdomain
- Author
-
Katarzyna Bebenek, Samuel H. Wilson, Thomas A. Darden, Thomas A. Kunkel, Hyeung-Rak Kim, William A. Beard, and José R. Casas-Finet
- Subjects
DNA polymerase ,Biochemistry ,Protein Structure, Secondary ,Frameshift mutation ,Protein structure ,Frameshift Mutation ,Molecular Biology ,DNA Primers ,Alanine ,chemistry.chemical_classification ,biology ,Circular Dichroism ,RNA-Directed DNA Polymerase ,Templates, Genetic ,Cell Biology ,Processivity ,HIV Reverse Transcriptase ,Reverse transcriptase ,Protein Structure, Tertiary ,Amino acid ,Spectrometry, Fluorescence ,chemistry ,Mutagenesis ,HIV-1 ,biology.protein ,Primer (molecular biology) ,Protein Processing, Post-Translational - Abstract
We have analyzed two human immunodeficiency virus (HIV-1) reverse transcriptase mutants of helix H in the thumb subdomain suggested by x-ray crystallography to interact with the primer strand of the template-primer. These enzymes, G262A and W266A, were previously shown to have greatly elevated dissociation rate constants for template-primer and to be much less sensitive to inhibition by 3'-azidodeoxythymidine 5'-triphosphate. Here we describe their processivity and error specificity. The results reveal that: (i) both enzymes have reduced processivity and lower fidelity for template-primer slippage errors, (ii) they differ from each other in sequence-dependent termination of processive synthesis and in error specificity, and (iii) the magnitude of the mutator effect relative to wild-type enzyme for deletions in homopolymeric sequences decreases as the length of the run increases. Thus amino acid substitutions in a subdomain thought to interact with the duplex template-primer confer a strand slippage mutator phenotype to a replicative DNA polymerase. This suggests that interactions between specific amino acids and the primer stem at positions well removed from the active site are critical determinants of processivity and fidelity. These effects, obtained in aqueous solution during catalytic cycling, are consistent with and support the existing crystallographic structural model.
- Published
- 1995
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