34 results on '"Replisome"'
Search Results
2. The eukaryotic replisome tolerates leading‐strand base damage by replicase switching
- Author
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Guilliam, Thomas A and Yeeles, Joseph TP
- Published
- 2021
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3. Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL
- Author
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Michael Anikin, Azadeh Sarfallah, Angelica Zamudio-Ochoa, and Dmitry Temiakov
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DNA Replication ,Models, Molecular ,Mitochondrial DNA ,POLRMT ,Molecular Conformation ,Replication Origin ,Biology ,DNA, Mitochondrial ,Primosome ,General Biochemistry, Genetics and Molecular Biology ,Mitochondrial Proteins ,Structure-Activity Relationship ,Transcription (biology) ,Molecular Biology ,Transcription Initiation, Genetic ,Polymerase ,Base Sequence ,General Immunology and Microbiology ,Mechanism (biology) ,General Neuroscience ,Articles ,Mitochondria ,Cell biology ,DNA-Binding Proteins ,biology.protein ,Nucleic Acid Conformation ,RNA ,Replisome ,Primer (molecular biology) - Abstract
The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.
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- 2021
- Full Text
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4. TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans
- Author
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Remi Sonneville, Tom D Deegan, Yisui Xia, Ryo Fujisawa, and Karim Labib
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DNA Replication ,CMG helicase ,CUL‐2LRR‐1 ,Synthetic lethality ,DNA-Directed DNA Polymerase ,General Biochemistry, Genetics and Molecular Biology ,Article ,Ubiquitin ,Multienzyme Complexes ,Animals ,CDC‐48 ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,UBXN‐3 ,General Immunology and Microbiology ,biology ,General Neuroscience ,Helicase ,Signal transducing adaptor protein ,DNA Replication, Repair & Recombination ,Post-translational Modifications, Proteolysis & Proteomics ,Articles ,biology.organism_classification ,Cullin Proteins ,TIMELESS‐TIPIN ,Ubiquitin ligase ,Cell biology ,DNA replication termination ,biology.protein ,Replisome ,Carrier Proteins ,Synthetic Lethal Mutations - Abstract
The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer., CUL2/LRR1‐dependent CMG helicase ubiquitination is induced by replisome‐associated TIM‐1/TIPN‐1 and further processed by the p97/CDC‐48 unfoldase stimulated by the UFD‐1, NPL‐4 and UBXN‐3 adaptor proteins.
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- 2021
5. Timeless couples G‐quadruplex detection with processing by DDX11 helicase during DNA replication
- Author
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Lerner, Leticia K, Holzer, Sandro, Kilkenny, Mairi L, Šviković, Saša, Murat, Pierre, Schiavone, Davide, Eldridge, Cara B, Bittleston, Alice, Maman, Joseph D, Branzei, Dana, Stott, Katherine, Pellegrini, Luca, and Sale, Julian E
- Published
- 2020
- Full Text
- View/download PDF
6. The eukaryotic replisome tolerates leading‐strand base damage by replicase switching
- Author
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Thomas A. Guilliam and Joseph Tp Yeeles
- Subjects
DNA Replication ,Saccharomyces cerevisiae Proteins ,DNA polymerase ,RNA-dependent RNA polymerase ,DNA-Directed DNA Polymerase ,Saccharomyces cerevisiae ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Nucleotide ,translesion synthesis ,Molecular Biology ,DNA damage tolerance ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,replisome ,General Immunology and Microbiology ,biology ,General Neuroscience ,DNA replication ,DNA Replication, Repair & Recombination ,Articles ,Yeast ,Cell biology ,chemistry ,biology.protein ,Replisome ,Thymine ,030217 neurology & neurosurgery ,DNA ,DNA Damage - Abstract
The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε., The common oxidative DNA base lesion thymine glycol can surprisingly be bypassed by reconstituted yeast replisomes in the absence of dedicated translesion synthesis polymerases.
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- 2021
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7. A Timeless Tale: G4 structure recognition by the fork protection complex triggers unwinding by <scp>DDX</scp> 11 helicase
- Author
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Catherine H. Freudenreich
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0303 health sciences ,General Immunology and Microbiology ,Timeless ,General Neuroscience ,Helicase ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Structure recognition ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,DDX11 ,Fork (system call) ,biology.protein ,Replisome ,Molecular Biology ,030217 neurology & neurosurgery ,DNA ,030304 developmental biology ,Binding domain - Abstract
How the replisome senses and deals with DNA secondary structures has been a mystery. A new study from the Sale and Pellegrini laboratories finds that the Timeless protein has a G-quadruplex binding domain that works together with the DDX11 helicase to facilitate replication of G4 DNA structures.
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- 2020
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8. Timeless couples G‐quadruplex detection with processing by <scp>DDX</scp> 11 helicase during <scp>DNA</scp> replication
- Author
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Joseph D. Maman, Cara B. Eldridge, Davide Schiavone, Julian E. Sale, Alice Bittleston, Luca Pellegrini, Sandro Holzer, Dana Branzei, Saša Šviković, Katherine Stott, Leticia K. Lerner, Mairi L. Kilkenny, Pierre Murat, MRC Laboratory of Molecular Biology [Cambridge, UK] (LMB), University of Cambridge [UK] (CAM)-Medical Research Council, Department of Biochemistry, University of Cambridge, and IFOM, Istituto FIRC di Oncologia Molecolare (IFOM)
- Subjects
DNA Replication ,fork protection complex ,Timeless ,DNA damage ,timeless ,Cell Cycle Proteins ,Biology ,G-quadruplex ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,DEAD-box RNA Helicases ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein Domains ,Structural Biology ,[CHIM]Chemical Sciences ,Humans ,News & Views ,Molecular Biology ,Recombination & Repair ,030304 developmental biology ,0303 health sciences ,replisome ,General Immunology and Microbiology ,DNA synthesis ,General Neuroscience ,DNA Helicases ,Intracellular Signaling Peptides and Proteins ,DNA replication ,DNA Replication, Repair & Recombination ,Helicase ,Articles ,Cell biology ,G‐quadruplex ,G-Quadruplexes ,chemistry ,biology.protein ,Replisome ,030217 neurology & neurosurgery ,DNA ,DNA Damage - Abstract
Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C‐terminal region a previously unappreciated DNA‐binding domain that exhibits specific binding to G‐quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4‐forming sequences, and exhibits partial redundancy with an adjacent PARP‐binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4‐forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication‐hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis., Cooperation of a helicase and a component of the fork protection complex allows eukaryotic replisomes to detect and respond to DNA secondary structures.
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- 2020
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9. Excessive excision of correct nucleotides during <scp>DNA</scp> synthesis explained by replication hurdles
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Smita S. Patel, Anupam Singh, Manjula Pandey, Divya Nandakumar, Kevin D. Raney, and Y. Whitney Yin
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DNA Replication ,primer shuttling ,Exonuclease ,DNA Repair ,DNA polymerase ,exonuclease activity ,translocation ,DNA Primase ,DNA-Directed DNA Polymerase ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bacteriophage T7 ,Catalytic Domain ,replication hurdles ,Molecular Biology ,Polymerase ,DNA Primers ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,biology ,DNA synthesis ,Nucleotides ,General Neuroscience ,DNA Replication, Repair & Recombination ,Helicase ,DNA ,Articles ,Cell biology ,Exodeoxyribonucleases ,chemistry ,Mutation ,biology.protein ,Proofreading ,Replisome ,030217 neurology & neurosurgery - Abstract
The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer‐end to the exonuclease site as a “cost of proofreading”. Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase–polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer‐ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active‐site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer‐ends from mutagenic extensions., Frequent shuttling of primer ends between polymerase‐ and exonuclease‐active sites may serve as proofreading mechanism preventing them from mutagenic extension.
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- 2020
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10. Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin
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Evrin, Cecile, Maman, Joseph D, Diamante, Aurora, Pellegrini, Luca, and Labib, Karim
- Published
- 2018
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11. The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion
- Author
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Ivanov, Miroslav P, Ladurner, Rene, Poser, Ina, Beveridge, Rebecca, Rampler, Evelyn, Hudecz, Otto, Novatchkova, Maria, Hériché, Jean‐Karim, Wutz, Gordana, van der Lelij, Petra, Kreidl, Emanuel, Hutchins, James RA, Axelsson‐Ekker, Heinz, Ellenberg, Jan, Hyman, Anthony A, Mechtler, Karl, and Peters, Jan‐Michael
- Published
- 2018
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12. Spatial separation between replisome‐ and template‐induced replication stress signaling
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Ronald P.C. Wong, Yasukazu Daigaku, Helle D. Ulrich, Néstor García-Rodríguez, and Magdalena E. Morawska
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DNA Replication ,0301 basic medicine ,Exonuclease ,Saccharomyces cerevisiae Proteins ,replication stress ,DNA damage bypass ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,Exo1 ,Article ,General Biochemistry, Genetics and Molecular Biology ,S Phase ,DNA damage checkpoint ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,DNA, Fungal ,Molecular Biology ,General Immunology and Microbiology ,Replication stress ,General Neuroscience ,postreplication repair ,DNA Replication, Repair & Recombination ,Cell Cycle Checkpoints ,Articles ,Replication (computing) ,Cell biology ,Exodeoxyribonucleases ,030104 developmental biology ,chemistry ,biology.protein ,Replisome ,030217 neurology & neurosurgery ,DNA - Abstract
Polymerase‐blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single‐stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re‐priming downstream of lesions can give rise to daughter‐strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9‐dependent mechanism of damage signaling is distinct from the Mrc1‐dependent, fork‐associated response to replication stress induced by conditions such as nucleotide depletion or replisome‐inherent problems, but reminiscent of replication‐independent checkpoint activation by single‐stranded DNA. Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase‐blocking lesions mainly emanates from Exo1‐processed, postreplicative daughter‐strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome‐ versus template‐induced checkpoint signaling.
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- 2018
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13. A MatP-divisome interaction coordinates chromosome segregation with cell division inE. coli
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Romain Borne, Frédéric Boccard, Axel Thiel, Pauline Dupaigne, Romain Mercier, Emmanuelle Gigant, and Olivier Espéli
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Genetics ,General Immunology and Microbiology ,Cell division ,General Neuroscience ,Ter protein ,food and beverages ,Chromosome ,Biology ,Origin of replication ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Chromosome segregation ,chemistry.chemical_compound ,chemistry ,medicine ,Replisome ,human activities ,Molecular Biology ,Escherichia coli ,DNA - Abstract
Initiation of chromosome segregation in bacteria is achieved by proteins acting near the origin of replication. Here, we report that the precise choreography of the terminus region of the Escherichia coli chromosome is also tightly controlled. The segregation of the terminus (Ter) macrodomain (MD) involves the structuring factor MatP. We characterized that migration of the Ter MD from the new pole to mid-cell and its subsequent persistent localization at mid-cell relies on several processes. First, the replication of the Ter DNA is concomitant with its recruitment from the new pole to mid-cell in a sequential order correlated with the position on the genetic map. Second, using a strain carrying a linear chromosome with the Ter MD split in two parts, we show that replisomes are repositioned at mid-cell when replication of the Ter occurs. Third, we demonstrate that anchoring the Ter MD at mid-cell depends on the specific interaction of MatP with the division apparatus-associated protein ZapB. Our results reveal how segregation of the Ter MD is integrated in the cell-cycle control.
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- 2012
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14. Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation
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Alberto Sanchez-Diaz, Karim Labib, Giacomo De Piccoli, Sugopa Sengupta, and Frederick van Deursen
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General Immunology and Microbiology ,General Neuroscience ,DNA replication ,Biology ,Pre-replication complex ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Replication factor C ,Minichromosome maintenance ,Control of chromosome duplication ,Replisome ,Origin recognition complex ,Molecular Biology ,S phase - Abstract
Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2-7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2-7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2-7 when cells entered S-phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45–MCM–GINS helicase at DNA replication origins.
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- 2012
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15. E. coli DNA replication in the absence of free beta clamps
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LEADING-STRAND ,LAGGING-STRAND SYNTHESIS ,single molecule ,DNA replication ,BACTERIOPHAGE-T4 PROTEINS ,fluorescence microscopy ,ESCHERICHIA-COLI ,SLIDING CLAMP ,FORK ,POLYMERASE-III HOLOENZYME ,clamp recycling ,REPLISOME ,SINGLE-MOLECULE ,PROCESSIVITY CLAMP - Abstract
During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the beta sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess beta clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps. The EMBO Journal (2011) 30, 1830-1840. doi:10.1038/emboj.2011.84; Published online 25 March 2011
- Published
- 2011
16. E. coliDNA replication in the absence of free β clamps
- Author
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Jack D. Griffith, Nicholas E. Dixon, Nathan A. Tanner, Gökhan Tolun, Antoine M. van Oijen, Joseph J. Loparo, and Slobodan Jergic
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DNA clamp ,General Immunology and Microbiology ,biology ,Okazaki fragments ,DNA polymerase ,General Neuroscience ,DNA replication ,Processivity ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,biology.protein ,Replisome ,Primase ,Molecular Biology ,Polymerase - Abstract
During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the β sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps.
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- 2011
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17. MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication
- Author
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Yilun Liu, Eminet A Feyissa, Patrick J. Rochette, Tina V Su, and Xiaohua Xu
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DNA Replication ,Cell Cycle Proteins ,Replication Origin ,Eukaryotic DNA replication ,Pre-replication complex ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,DNA replication factor CDT1 ,Replication factor C ,Control of chromosome duplication ,Minichromosome maintenance ,Animals ,Humans ,Molecular Biology ,Genetics ,Minichromosome Maintenance Proteins ,RecQ Helicases ,General Immunology and Microbiology ,biology ,General Neuroscience ,Cell Cycle ,Nuclear Proteins ,Minichromosome Maintenance Complex Component 2 ,DNA ,Minichromosome Maintenance Complex Component 7 ,Chromatin ,DNA-Binding Proteins ,biology.protein ,Replisome ,Origin recognition complex - Abstract
Mutations in RECQ4, a member of the RecQ family of DNA helicases, have been linked to the progeroid disease Rothmund-Thomson Syndrome. Attempts to understand the complex phenotypes observed in recq4-deficient cells suggest a potential involvement in DNA repair and replication, yet the molecular basis of the function of RECQ4 in these processes remains unknown. Here, we report the identification of a highly purified chromatin-bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2-7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Importantly, complex formation and the association of RECQ4 with the replication origin are cell-cycle regulated. Furthermore, we show that MCM10 is essential for the integrity of the RECQ4-MCM replicative helicase complex. MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity, and that this interaction may be modulated by cyclin-dependent kinase phosphorylation. Thus, these studies show that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells.
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- 2009
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18. Mechanism of polymerase collision release from sliding clamps on the lagging strand
- Author
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Georgescu, Roxana E, Kurth, Isabel, Yao, Nina Y, Stewart, Jelena, Yurieva, Olga, and O'Donnell, Mike
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- 2009
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19. Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance
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Jennifer A. Cobb, Susan M. Gasser, Lotte Bjergbaek, and Monica Tsai‐Pflugfelder
- Subjects
DNA Replication ,Saccharomyces cerevisiae Proteins ,Cell cycle checkpoint ,DNA polymerase ,RecQ helicase ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,Article ,General Biochemistry, Genetics and Molecular Biology ,Minichromosome maintenance ,Two-Hybrid System Techniques ,Hydroxyurea ,Immunoprecipitation ,Phosphorylation ,Molecular Biology ,Genetics ,RecQ Helicases ,General Immunology and Microbiology ,biology ,General Neuroscience ,Cell Cycle ,DNA Helicases ,Helicase ,G2-M DNA damage checkpoint ,Checkpoint Kinase 2 ,DNA Topoisomerases, Type I ,biology.protein ,Replisome ,Sgs1 - Abstract
The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.
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- 2004
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20. DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1
- Author
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Kenji Shimada, Susan M. Gasser, Jennifer A. Cobb, Christian Frei, and Lotte Bjergbaek
- Subjects
DNA re-replication ,Saccharomyces cerevisiae Proteins ,Time Factors ,DNA Repair ,Replication Origin ,Eukaryotic DNA replication ,DNA-Directed DNA Polymerase ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Biology ,Pre-replication complex ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,S Phase ,Control of chromosome duplication ,Hydroxyurea ,Point Mutation ,DNA, Fungal ,Molecular Biology ,S phase ,Nucleic Acid Synthesis Inhibitors ,Genetics ,RecQ Helicases ,General Immunology and Microbiology ,General Neuroscience ,DNA Helicases ,Intracellular Signaling Peptides and Proteins ,DNA replication ,Articles ,Protein Structure, Tertiary ,Kinetics ,Replisome ,Origin recognition complex - Abstract
To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.
- Published
- 2003
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21. Building a double hexamer of DNA helicase at eukaryotic replication origins
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Karim Labib
- Subjects
Genetics ,General Immunology and Microbiology ,General Neuroscience ,Eukaryotic DNA replication ,Biology ,Pre-replication complex ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Minichromosome maintenance ,Control of chromosome duplication ,Replisome ,Origin recognition complex ,Primase ,Molecular Biology ,Replication protein A - Abstract
The Mcm2‐7 complex, the catalytic core of the eukaryotic replicative DNA helicase, undergoes a complex series of transformations at origins of DNA replication. During G1 phase, it is loaded around double‐strand DNA at origins as an inactive double hexamer, which is subsequently remodelled in situ to activate the helicase during S phase. Work in this issue of The EMBO Journal sheds light on the role of the Cdt1 protein in generating Mcm2‐7 double hexamers during the loading reaction.
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- 2011
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22. And-1 coordinates with Claspin for efficient Chk1 activation in response to replication stress
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Michael G. Kemp, Wenge Zhu, Melvin L. DePamphilis, Jing Hao, Zhiyong Han, Christelle de Renty, Yongming Li, and Haijie Xiao
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DNA Replication ,genetic processes ,Regulator ,Fluorescent Antibody Technique ,Eukaryotic DNA replication ,Biology ,Pre-replication complex ,environment and public health ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Antibodies ,Mass Spectrometry ,Replication factor C ,Humans ,Immunoprecipitation ,Phosphorylation ,RNA, Small Interfering ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,General Immunology and Microbiology ,General Neuroscience ,Articles ,Cell biology ,DNA replication checkpoint ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,HEK293 Cells ,Checkpoint Kinase 1 ,Replisome ,Origin recognition complex ,RNA Interference ,biological phenomena, cell phenomena, and immunity ,Protein Kinases - Abstract
The replisome is important for DNA replication checkpoint activation, but how specific components of the replisome coordinate with ATR to activate Chk1 in human cells remains largely unknown. Here, we demonstrate that And-1, a replisome component, acts together with ATR to activate Chk1. And-1 is phosphorylated at T826 by ATR following replication stress, and this phosphorylation is required for And-1 to accumulate at the damage sites, where And-1 promotes the interaction between Claspin and Chk1, thereby stimulating efficient Chk1 activation by ATR. Significantly, And-1 binds directly to ssDNA and facilitates the association of Claspin with ssDNA. Furthermore, And-1 associates with replication forks and is required for the recovery of stalled forks. These studies establish a novel ATR–And-1 axis as an important regulator for efficient Chk1 activation and reveal a novel mechanism of how the replisome regulates the replication checkpoint and genomic stability.
- Published
- 2014
23. Replication slippage involves DNA polymerase pausing and dissociation
- Author
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Danielle Canceill, Enrique Viguera, S. Dusko Ehrlich, Unité de recherche Génétique Microbienne (UGM), and Institut National de la Recherche Agronomique (INRA)
- Subjects
DNA Replication ,DNA polymerase II ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,DNA-Directed DNA Polymerase ,DNA polymerase delta ,Article ,General Biochemistry, Genetics and Molecular Biology ,Viral Proteins ,Replication slippage ,Control of chromosome duplication ,ADN POLYMERASE ,Molecular Biology ,DNA Polymerase III ,Repetitive Sequences, Nucleic Acid ,Recombination, Genetic ,Genetics ,Binding Sites ,DNA clamp ,Models, Genetic ,General Immunology and Microbiology ,biology ,General Neuroscience ,DNA replication ,Cell biology ,biology.protein ,Replisome ,DNA polymerase I - Abstract
Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.
- Published
- 2001
- Full Text
- View/download PDF
24. Impairment of lagging strand synthesis triggers the formation of a RuvABC substrate at replication forks
- Author
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Hélène Bierne, Maria-Jose Flores, Bénédicte Michel, S. Dusko Ehrlich, Unité de recherche Génétique Microbienne (UGM), Institut National de la Recherche Agronomique (INRA), and ProdInra, Migration
- Subjects
DNA Replication ,Replication fork reversal ,DNA Repair ,DNA repair ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity ,03 medical and health sciences ,RuvABC ,Bacterial Proteins ,ADN POLYMERASE ,Escherichia coli ,Holliday junction ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,DNA Polymerase III ,DNA Primers ,030304 developmental biology ,Recombination, Genetic ,Genetics ,RecBCD ,0303 health sciences ,Endodeoxyribonucleases ,Base Sequence ,General Immunology and Microbiology ,030306 microbiology ,Escherichia coli Proteins ,General Neuroscience ,DNA Helicases ,DNA replication ,DNA-Binding Proteins ,Rec A Recombinases ,Phenotype ,Genes, Bacterial ,Mutation ,bacteria ,Replisome ,Homologous recombination - Abstract
The holD gene codes for the psi subunit of the Escherichia coli DNA polymerase III holoenzyme, a component of the gamma complex clamp loader. A holD mutant was isolated for the first time in a screen for mutations that increase the frequency of tandem repeat deletions. In contrast to tandem repeat deletions in wild-type strains, deletion events stimulated by the holD mutation require RecA. They do not require RecF, and hence do not result from the recombinational repair of gaps, arguing against uncoupling of the leading and lagging strand polymerases in the holD mutant. The holD recBC combination of mutations is lethal and holD recBts recCts strains suffer DNA double-strand breaks (DSBs) at restrictive temperature. DSBs require the presence of the Holliday junction-specific enzymes RuvABC and are prevented in the presence of RecBCD. We propose that impairment of replication due to the holD mutation causes the arrest of the entire replisome; consequently, Holliday junctions are formed by replication fork reversal, and unequal crossing over during RecA- and RecBCD-mediated re-incorporation of reversed forks causes the hyper-recombination phenotype.
- Published
- 2001
- Full Text
- View/download PDF
25. A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein
- Author
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Peter Ahnert, Kristen Moore Picha, and Smita S. Patel
- Subjects
Base Sequence ,General Immunology and Microbiology ,General Neuroscience ,DNA, Single-Stranded ,Articles ,DNA Primase ,Biology ,Pre-replication complex ,RNA Helicase A ,Primosome ,General Biochemistry, Genetics and Molecular Biology ,DnaA ,Kinetics ,Biochemistry ,Prokaryotic DNA replication ,Bacteriophage T7 ,DNA, Viral ,Biophysics ,Nucleic Acid Conformation ,Replisome ,Primase ,Molecular Biology ,Replication protein A ,Protein Binding - Abstract
We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.
- Published
- 2000
- Full Text
- View/download PDF
26. A moving DNA replication factory in Caulobacter crescentus
- Author
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Jensen, Rasmus B., Wang, Sherry C., and Shapiro, Lucy
- Published
- 2001
- Full Text
- View/download PDF
27. Devoted to the lagging strand_the chi subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly
- Author
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Mike O'Donnell, Alexander Yuzhakov, Zvi Kelman, and Jelena Andjelkovic
- Subjects
DNA polymerase ,Coenzymes ,Peptide Chain Elongation, Translational ,DNA, Single-Stranded ,dnaN ,Sodium Chloride ,DNA polymerase delta ,General Biochemistry, Genetics and Molecular Biology ,DNA polymerase III holoenzyme ,Molecular Biology ,DNA Polymerase III ,Binding Sites ,DNA clamp ,General Immunology and Microbiology ,biology ,General Neuroscience ,Osmolar Concentration ,DNA ,Processivity ,DNA-Binding Proteins ,stomatognathic diseases ,Biochemistry ,biology.protein ,Biophysics ,Replisome ,DNA polymerase I ,Research Article - Abstract
Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA. We examine here the function of the psi subunit of the gamma complex clamp loader. Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi. These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.
- Published
- 1998
- Full Text
- View/download PDF
28. Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?
- Author
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Richard P. Novick, Maria-Elena Fernandez-Beros, and Ruzhong Jin
- Subjects
DNA Replication ,DNA, Bacterial ,Staphylococcus aureus ,DNA Ligases ,Inverted repeat ,Molecular Sequence Data ,DNA, Single-Stranded ,Replication Origin ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Plasmid ,Bacterial Proteins ,Recognition sequence ,Molecular Biology ,Electrophoresis, Agar Gel ,Base Sequence ,General Immunology and Microbiology ,DNA, Superhelical ,General Neuroscience ,DNA replication ,Nicking enzyme ,Molecular biology ,DNA-Binding Proteins ,Oligodeoxyribonucleotides ,Cruciform ,Rolling circle replication ,Biophysics ,Nucleic Acid Conformation ,Replisome ,Electrophoresis, Polyacrylamide Gel ,DNA, Circular ,Plasmids ,Research Article - Abstract
pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter.
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- 1997
- Full Text
- View/download PDF
29. The φX174‐type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition
- Author
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Jones, Jessica M. and Nakai, Hiroshi
- Published
- 1997
- Full Text
- View/download PDF
30. SCF(Dia2) regulates DNA replication forks during S-phase in budding yeast
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Takumi Kamura, Makiko Komata, Katsuhiko Shirahige, Satoru Mimura, and Tsutomu Kishi
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Repetitive Sequences, Amino Acid ,Saccharomyces cerevisiae Proteins ,DNA damage ,Chromosomal Proteins, Non-Histone ,Saccharomyces cerevisiae ,Amino Acid Motifs ,Cell Cycle Proteins ,F-box protein ,DNA-binding protein ,General Biochemistry, Genetics and Molecular Biology ,Article ,S Phase ,Leucine ,Cell Cycle Protein ,Molecular Biology ,Genetics ,General Immunology and Microbiology ,biology ,DNA, Superhelical ,Protein Stability ,General Neuroscience ,F-Box Proteins ,DNA replication ,biology.organism_classification ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Tetratricopeptide ,biology.protein ,Replisome - Abstract
Dia2 is an F-box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae. The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two-hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2-binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCF(Dia2) both in vivo and in vitro. Domain analysis of Dia2 revealed that the leucine-rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP-on-chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.
- Published
- 2009
31. Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli
- Author
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Kirsten Skarstad, Elliott Crooke, and Solveig Fossum
- Subjects
DNA Replication ,DNA, Bacterial ,Mutant ,Replication Origin ,Biology ,medicine.disease_cause ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Article ,chemistry.chemical_compound ,Plasmid ,SeqA protein domain ,Bacterial Proteins ,medicine ,Escherichia coli ,Molecular Biology ,Genetics ,Mutation ,General Immunology and Microbiology ,General Neuroscience ,Escherichia coli Proteins ,Cell Cycle ,DNA replication ,DNA Restriction Enzymes ,Cell cycle ,DNA-Binding Proteins ,chemistry ,Bromodeoxyuridine ,Microscopy, Fluorescence ,Replisome ,DNA ,Bacterial Outer Membrane Proteins ,Plasmids - Abstract
The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.1-0.2 generations. The role of origin inactivation (sequestration) by the SeqA protein in origin colocalization was studied by comparing sequestration-deficient mutants with wild-type cells. Cells with mutant, non-sequesterable origins showed wild-type colocalization of sister origins. In contrast, cells unable to sequester new origins due to loss of SeqA, showed aberrant localization of origins indicating a lack of organization of new origins. In these cells, aberrant replisome organization was also found. These results suggest that correct organization of sister origins and sister replisomes is dependent on the binding of SeqA protein to newly formed DNA at the replication forks, but independent of origin sequestration. In agreement, in vitro experiments indicate that SeqA is capable of pairing newly replicated DNA molecules.
- Published
- 2007
32. A moving DNA replication factory in Caulobacter crescentus
- Author
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Sherry C. Wang, Lucy Shapiro, and Rasmus B. Jensen
- Subjects
DNA Replication ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,Replication Origin ,Pre-replication complex ,General Biochemistry, Genetics and Molecular Biology ,Article ,Minichromosome maintenance ,Control of chromosome duplication ,Bacterial Proteins ,Caulobacter crescentus ,Molecular Biology ,dnaB helicase ,Genetics ,General Immunology and Microbiology ,biology ,Models, Genetic ,General Neuroscience ,Cell Cycle ,DNA replication ,DNA Helicases ,biology.organism_classification ,Kinetics ,Luminescent Proteins ,Origin recognition complex ,Replisome ,Chromosomes, Fungal ,DnaB Helicases ,Cell Division - Abstract
The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.
- Published
- 2001
33. Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site
- Author
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Joonsoo Lee, Takahiro Kusakabe, Khandan Baradaran, and Charles C. Richardson
- Subjects
DNA polymerase II ,Molecular Sequence Data ,DNA, Single-Stranded ,DNA Primase ,Primosome ,General Biochemistry, Genetics and Molecular Biology ,DnaG ,Bacteriophage T7 ,Thymine Nucleotides ,Molecular Biology ,DNA Primers ,Oligoribonucleotides ,General Immunology and Microbiology ,biology ,Base Sequence ,General Neuroscience ,Circular bacterial chromosome ,DNA replication ,DNA Helicases ,Molecular biology ,Biochemistry ,DNA, Viral ,biology.protein ,Replisome ,Primase ,Primer (molecular biology) ,DNA, Circular ,Protein Binding ,Research Article - Abstract
The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.
- Published
- 1998
34. Are single-stranded circles intermediates in plasmid DNA replication?
- Author
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H te Riele, Stanislav Dusko Ehrlich, B Michel, Unité de recherche Génétique Microbienne (UGM), and Institut National de la Recherche Agronomique (INRA)
- Subjects
DNA Replication ,Staphylococcus aureus ,DNA polymerase II ,DNA, Single-Stranded ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,Coliphages ,Primosome ,General Biochemistry, Genetics and Molecular Biology ,Control of chromosome duplication ,Escherichia coli ,Molecular Biology ,Plasmid preparation ,DNA clamp ,General Immunology and Microbiology ,General Neuroscience ,DNA replication ,Nucleic Acid Hybridization ,DNA Restriction Enzymes ,Molecular biology ,Microscopy, Electron ,Protein Biosynthesis ,biology.protein ,Replisome ,DNA supercoil ,DNA, Circular ,Bacillus subtilis ,Plasmids ,Research Article - Abstract
Plasmid pC194 exists as circular double-stranded and single-stranded DNA in Bacillus subtilis and Staphylococcus aureus. We report here that the plasmid pHV33, composed of pBR322 and pC194, exists as double- and single-stranded DNA in Escherichia coli, provided that the replication functions of pC194 are intact. Single-stranded pHV33 DNA is converted to double-stranded DNA by complementary strand synthesis probably initiated at rriB, a primosome assembly site present on pBR322. The efficiency of complementary strand synthesis affects the double-stranded copy number, which suggests that single-stranded DNA is a plasmid replication intermediate.
- Published
- 1986
- Full Text
- View/download PDF
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