31 results on '"Rakhi Rajan"'
Search Results
2. The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies
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Sydney Newsom, Hari Priya Parameshwaran, Lindsie Martin, and Rakhi Rajan
- Subjects
clustered regularly interspaced short palindromic repeats and clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) ,adaptive immunity ,Cas9 ,cascade ,gene editing ,bacterial pathogenesis ,Microbiology ,QR1-502 - Abstract
Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine.
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- 2021
- Full Text
- View/download PDF
3. RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a
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Ramya Sundaresan, Hari Priya Parameshwaran, S.D. Yogesha, Mark Walter Keilbarth, and Rakhi Rajan
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CRISPR ,Cas9 endonucleases ,Cpf1 ,Cas12a ,Francisella tularensis novicida ,RNA-independent DNA cleavage ,Mn2+-specific CRISPR activity ,SpyCas9 ,FnoCas9 ,FnoCas12a ,Biology (General) ,QH301-705.5 - Abstract
CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications.
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- 2017
- Full Text
- View/download PDF
4. Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
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Zhicheng Zuo, Ashwini Zolekar, Kesavan Babu, Victor JT Lin, Hamed S Hayatshahi, Rakhi Rajan, Yu-Chieh Wang, and Jin Liu
- Subjects
HEK293T cell ,CRISPR-Cas9 ,catalysis model ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.
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- 2019
- Full Text
- View/download PDF
5. Conserved DNA motifs in the type II-A CRISPR leader region
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Mason J. Van Orden, Peter Klein, Kesavan Babu, Fares Z. Najar, and Rakhi Rajan
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CRISPR-Cas systems ,Type II-A CRISPR ,Leader-repeat ,Adaptation ,Medicine ,Biology (General) ,QH301-705.5 - Abstract
The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.
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- 2017
- Full Text
- View/download PDF
6. Optimized protocols for the characterization of Cas12a activities
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Lindsie Martin, Saadi Rostami, and Rakhi Rajan
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- 2023
7. Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence
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Pratibha Kumari, Peter Z. Qin, Xiongping Chen, Kesavan Babu, Venkatesan Kathiresan, Rakhi Rajan, Sydney Newsom, Jin Liu, and Hari Priya Parameshwaran
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Protein Conformation, alpha-Helical ,Streptococcus pyogenes ,Gene Expression ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Cleavage (embryo) ,Biochemistry ,DNA sequencing ,Substrate Specificity ,chemistry.chemical_compound ,Protein Domains ,CRISPR-Associated Protein 9 ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Protein Interaction Domains and Motifs ,Guide RNA ,DNA Cleavage ,Nuclease ,Binding Sites ,biology ,Chemistry ,Cas9 ,RNA ,DNA ,Kinetics ,Mutation ,Biophysics ,biology.protein ,Thermodynamics ,Protein Conformation, beta-Strand ,CRISPR-Cas Systems ,Protein Binding ,RNA, Guide, Kinetoplastida - Abstract
CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.
- Published
- 2021
8. Molecular Details of DNA Integration by CRISPR-Associated Proteins During Adaptation in Bacteria and Archaea
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Tamara, Flusche and Rakhi, Rajan
- Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in bacteria and archaea, where immunological memory is retained in the CRISPR locus as short pieces of the intruding nucleic acid, termed spacers. The adaptation to new infections occurs through the integration of a new spacer into the CRISPR array. For immune protection, spacers are transcribed into CRISPR RNAs (crRNA) that are used to guide the effector nuclease of the system in sequence-dependent target cleavage. Spacers originate as a prespacer from either DNA or RNA depending on the CRISPR-Cas system being observed, and the nearly universal Cas proteins, Cas1 and Cas2, insert the prespacer into the CRISPR locus during adaptation in all systems that contain them. The mechanism of site-specific prespacer integration varies across CRISPR classes and types, and distinct differences can even be found within the same subtype. In this review, the current knowledge on the mechanisms of prespacer integration in type II-A CRISPR-Cas systems will be described. Comparisons of the currently characterized type II-A systems show that distinct mechanisms exist within different members of this subtype and are correlated to sequence-specific interactions of Cas proteins and the DNA elements present in the CRISPR array. These observations indicate that nature has fine-tuned the mechanistic details while performing the basic step of DNA integration by Cas proteins, which offers unique advantages to develop Cas1-Cas2-based biotechnology.
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- 2022
9. The bridge helix of Cas12a imparts selectivity in cis ‐DNA cleavage and regulates trans ‐DNA cleavage
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Peter Z. Qin, Venkatesan Kathiresan, Kesavan Babu, Christine Tran, Hari Priya Parameshwaran, Kevin Guan, Rakhi Rajan, and Aleique Allen
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Stereochemistry ,CRISPR-Associated Proteins ,Biophysics ,DNA, Single-Stranded ,medicine.disease_cause ,Biochemistry ,Article ,03 medical and health sciences ,Bacterial Proteins ,Genome editing ,Structural Biology ,CRISPR-Associated Protein 9 ,Genetics ,medicine ,Deoxyribonuclease I ,CRISPR ,Francisella novicida ,DNA Cleavage ,Francisella ,Molecular Biology ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Endodeoxyribonucleases ,Chemistry ,Cas9 ,030302 biochemistry & molecular biology ,Cell Biology ,Helix ,Streptococcus pyogenes ,Nucleic Acid Conformation ,DNA supercoil ,CRISPR-Cas Systems ,Selectivity ,RNA, Guide, Kinetoplastida - Abstract
Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12a(KD2P)) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12a(KD2P) accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a(KD2P) also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.
- Published
- 2021
10. Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects
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Zhicheng Zuo, Kesavan Babu, Chhandosee Ganguly, Ashwini Zolekar, Sydney Newsom, Rakhi Rajan, Yu-Chieh Wang, and Jin Liu
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Gene Editing ,Genetics ,Humans ,RNA ,CRISPR-Cas Systems ,DNA Cleavage ,Endonucleases ,Research Articles ,Biotechnology - Abstract
The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.
- Published
- 2022
11. CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion
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Mason J Van Orden, Rakhi Rajan, and Sydney Newsom
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0301 basic medicine ,CRISPR-Associated Proteins ,Computational biology ,Microbiology ,Biochemistry ,DNA sequencing ,Insert (molecular biology) ,03 medical and health sciences ,chemistry.chemical_compound ,CRISPR ,Protein–DNA interaction ,Molecular Biology ,Phylogeny ,Sequence (medicine) ,Base Sequence ,030102 biochemistry & molecular biology ,biology ,DNA ,Cell Biology ,Integrase ,030104 developmental biology ,chemistry ,Nucleic acid ,biology.protein ,CRISPR-Cas Systems ,Protein Binding - Abstract
CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader–repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3′ end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader–repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1–Cas2 varied, with G1 stringently requiring a 5-nucleotide 3′ overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein–DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1–Cas2 toolset for inserting small DNAs into diverse DNA targets.
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- 2020
12. Molecular Details of DNA Integration by CRISPR-Associated Proteins During Adaptation in Bacteria and Archaea
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Tamara Flusche and Rakhi Rajan
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- 2022
13. Crystal structures of an unmodified bacterial tRNA reveal intrinsic structural flexibility and plasticity as general properties of unbound tRNAs
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Deanna Badong, Rakhi Rajan, Alfonso Mondragón, and Clarence W. Chan
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Crystallography ,Flexibility (anatomy) ,RNA ,Crystal structure ,RNA, Transfer, Amino Acyl ,Biology ,Ribosome ,Article ,medicine.anatomical_structure ,RNA, Transfer ,Transfer RNA ,Anticodon ,Escherichia coli ,Protein biosynthesis ,medicine ,Biophysics ,Nucleic Acid Conformation ,Molecule ,Nucleotide Motifs ,Structural motif ,Ribosomes ,Molecular Biology - Abstract
Ubiquitous across all domains of life, tRNAs constitute an essential component of cellular physiology, carry out an indispensable role in protein synthesis, and have been historically the subject of a wide range of biochemical and biophysical studies as prototypical folded RNA molecules. Although conformational flexibility is a well-established characteristic of tRNA structure, it is typically regarded as an adaptive property exhibited in response to an inducing event, such as the binding of a tRNA synthetase or the accommodation of an aminoacyl-tRNA into the ribosome. In this study, we present crystallographic data of a tRNA molecule to expand on this paradigm by showing that structural flexibility and plasticity are intrinsic properties of tRNAs, apparent even in the absence of other factors. Based on two closely related conformations observed within the same crystal, we posit that unbound tRNAs by themselves are flexible and dynamic molecules. Furthermore, we demonstrate that the formation of the T-loop conformation by the tRNA TΨC stem–loop, a well-characterized and classic RNA structural motif, is possible even in the absence of important interactions observed in fully folded tRNAs.
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- 2019
14. RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a
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Hari Priya Parameshwaran, S. D. Yogesha, Mark Walter Keilbarth, Ramya Sundaresan, and Rakhi Rajan
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0301 basic medicine ,Francisella tularensis novicida ,Time Factors ,CRISPR-Associated Proteins ,SpyCas9 ,General Biochemistry, Genetics and Molecular Biology ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Plasmid ,Cpf1 ,CRISPR-Associated Protein 9 ,Catalytic Domain ,CRISPR ,Deoxyribonuclease I ,Trypsin ,Cas9 endonucleases ,Guide RNA ,DNA Cleavage ,lcsh:QH301-705.5 ,Manganese ,Base Sequence ,Cas12a ,Chemistry ,Cas9 ,RNA ,DNA ,RNA-independent DNA cleavage ,Protospacer adjacent motif ,Kinetics ,030104 developmental biology ,Biochemistry ,lcsh:Biology (General) ,FnoCas9 ,Proteolysis ,FnoCas12a ,Nucleic Acid Conformation ,Mobile genetic elements ,Mn2+-specific CRISPR activity - Abstract
SUMMARY CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications, In Brief CRISPR-mediated gene editing involves DNA targeting using complementary guide RNAs (gRNAs). Sundaresan et al. find that Cas9/Cas12a orthologs cause RNA-independent, non-sequence-specific DNA cleavage in the presence of Mn2+ ions. These observations suggest that the type of Cas9/Cas12a and levels of intracellular metal ions may affect CRISPR-based genome editing.
- Published
- 2017
15. The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit
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Ramya Sundaresan, Dipali G. Sashital, Rakhi Rajan, Karthik Murugan, and Kesavan Babu
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Models, Molecular ,0301 basic medicine ,Transcription, Genetic ,Protein Conformation ,Computational biology ,Biology ,Diagnostic tools ,Article ,03 medical and health sciences ,Endonuclease ,Bacterial Proteins ,Protein Domains ,Genome editing ,Humans ,CRISPR ,Bacteriophages ,Clustered Regularly Interspaced Short Palindromic Repeats ,Guide RNA ,Molecular Biology ,Gene Editing ,Genetics ,Genome ,Bacteria ,Cas9 ,Cell Biology ,Endonucleases ,eye diseases ,humanities ,030104 developmental biology ,embryonic structures ,biology.protein ,CRISPR-Cas Systems ,Mobile genetic elements ,Genetic Engineering ,human activities ,RNA, Guide, Kinetoplastida - Abstract
CRISPR–Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR–Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR–Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR–Cas systems. This diversity has enabled further expansion of CRISPR–Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR–Cas systems.
- Published
- 2017
16. Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
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Kesavan Babu, Hamed S. Hayatshahi, Rakhi Rajan, Zhicheng Zuo, Yu-Chieh Wang, Victor Lin, Jin Liu, and Ashwini Zolekar
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0301 basic medicine ,QH301-705.5 ,Science ,Computational biology ,Cleavage (embryo) ,HEK293T cell ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Endonuclease ,0103 physical sciences ,Catalytic triad ,CRISPR ,Biology (General) ,Nuclease ,010304 chemical physics ,General Immunology and Microbiology ,biology ,Cas9 ,General Neuroscience ,General Medicine ,catalysis model ,3. Good health ,030104 developmental biology ,chemistry ,Structural biology ,biology.protein ,Medicine ,CRISPR-Cas9 ,DNA - Abstract
The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.
- Published
- 2019
17. Author response: Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
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Jin Liu, Ashwini Zolekar, Zhicheng Zuo, Victor Lin, Kesavan Babu, Rakhi Rajan, Yu-Chieh Wang, and Hamed S. Hayatshahi
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Nuclease ,biology ,Cas9 ,Stereochemistry ,Chemistry ,Streptococcus pyogenes ,biology.protein ,medicine ,State (functional analysis) ,medicine.disease_cause ,Domain (software engineering) - Published
- 2019
18. Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance
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Peter Z. Qin, Wei Jiang, S. D. Yogesha, Richard H. Nguyen, Nadia Amrani, Rakhi Rajan, and Kesavan Babu
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Models, Molecular ,Proline ,Mutation, Missense ,Computational biology ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,Article ,Genome engineering ,chemistry.chemical_compound ,Protein structure ,Bacterial Proteins ,CRISPR-Associated Protein 9 ,medicine ,CRISPR ,DNA Cleavage ,Gene Editing ,Mutation ,Chemistry ,Effector ,Cas9 ,RNA ,DNA ,Nucleic Acid Conformation ,CRISPR-Cas Systems ,RNA, Guide, Kinetoplastida - Abstract
CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.
- Published
- 2019
19. Nucleic Acid-Dependent Conformational Changes in CRISPR–Cas9 Revealed by Site-Directed Spin Labeling
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Rakhi Rajan, Peter Z. Qin, Richard H. Nguyen, Xiaojun Zhang, S. D. Yogesha, Carolina Reyes, and Narin S. Tangprasertchai
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Models, Molecular ,0301 basic medicine ,030103 biophysics ,Conformational change ,Streptococcus pyogenes ,Protein domain ,Biophysics ,Gene Expression ,Biology ,Biochemistry ,Protein Structure, Secondary ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Protein Domains ,CRISPR-Associated Protein 9 ,Escherichia coli ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Cloning, Molecular ,Staining and Labeling ,Cas9 ,Electron Spin Resonance Spectroscopy ,RNA ,DNA ,Cell Biology ,General Medicine ,Site-directed spin labeling ,Endonucleases ,Recombinant Proteins ,030104 developmental biology ,chemistry ,Nucleic acid ,Nitrogen Oxides ,Spin Labels ,CRISPR-Cas Systems ,Protein Binding ,RNA, Guide, Kinetoplastida - Abstract
In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.
- Published
- 2016
20. DNase H Activity of Neisseria meningitidis Cas9
- Author
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Yan Zhang, Rakhi Rajan, H. Steven Seifert, Alfonso Mondragón, and Erik J. Sontheimer
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DNA, Bacterial ,Molecular Sequence Data ,DNA, Single-Stranded ,Neisseria meningitidis ,Cleavage (embryo) ,medicine.disease_cause ,Article ,Genome engineering ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bacterial Proteins ,Cleave ,medicine ,Clustered Regularly Interspaced Short Palindromic Repeats ,Nucleotide Motifs ,Endodeoxyribonucleases ,Molecular Biology ,030304 developmental biology ,Genetics ,Trans-activating crRNA ,0303 health sciences ,biology ,Cas9 ,Cell Biology ,Biochemistry ,chemistry ,biology.protein ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,DNA ,RNA, Guide, Kinetoplastida - Abstract
Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs [CRISPR RNA (crRNA) and tracrRNA] are required for Cas9’s targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologues [including Neisseria meningitidis Cas9 (NmeCas9)] have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA-guided but PAM- and tracrRNA-independent. Beyond the need for guide-target pairing, this “DNase H” activity has no apparent sequence requirements, and the cleavage sites are measured from the 5′ end of the DNA substrate’s RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.
- Published
- 2015
21. Bridge Helix of Cas9 Impacts Target DNA Cleavage
- Author
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Rakhi Rajan
- Subjects
Materials science ,Dna cleavage ,Stereochemistry ,Helix ,Biophysics ,Bridge (interpersonal) - Published
- 2019
22. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site
- Author
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Alfonso Mondragón, Rakhi Rajan, and Amy K. Osterman
- Subjects
0301 basic medicine ,Models, Molecular ,030102 biochemistry & molecular biology ,biology ,DNA repair ,Topoisomerase ,Active site ,Isomerase ,Euryarchaeota ,Lyase ,AP lyase activity ,Protein Structure, Tertiary ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,DNA Topoisomerases, Type I ,Structural Biology ,Catalytic Domain ,Methanopyrus kandleri ,Genetics ,biology.protein ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Topoisomerase V - Abstract
Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.
- Published
- 2016
23. Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies
- Author
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Samuel H. Wilson, Rajendra Prasad, Bhupesh Taneja, Alfonso Mondragón, and Rakhi Rajan
- Subjects
Models, Molecular ,DNA repair ,Molecular Sequence Data ,Biology ,AP endonuclease ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Structural Biology ,Catalytic Domain ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Genetics ,AP site ,Amino Acid Sequence ,Lyase activity ,030304 developmental biology ,0303 health sciences ,Endodeoxyribonucleases ,Topoisomerase ,DNA ,Lyase ,DNA-(apurinic or apyrimidinic site) lyase ,Molecular biology ,DNA Topoisomerases, Type I ,chemistry ,Biochemistry ,biology.protein ,030217 neurology & neurosurgery - Abstract
Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)(2)] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)(2) domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.
- Published
- 2012
24. Structures of Minimal Catalytic Fragments of Topoisomerase V Reveals Conformational Changes Relevant for DNA Binding
- Author
-
Rakhi Rajan, Alfonso Mondragón, and Bhupesh Taneja
- Subjects
Models, Molecular ,Materials science ,PROTEINS ,Protein Conformation ,DNA repair ,Stereochemistry ,Article ,Phosphates ,chemistry.chemical_compound ,Type I topoisomerase ,Protein structure ,Structural Biology ,Catalytic Domain ,Binding site ,Molecular Biology ,Binding Sites ,Crystallography ,biology ,Topoisomerase ,Helix-Loop-Helix Motifs ,Active site ,DNA ,DNA Topoisomerases, Type I ,chemistry ,Helix ,biology.protein - Abstract
SummaryTopoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.
- Published
- 2010
25. Design and Synthesis of Substrate and Intermediate Analogue Inhibitors of S-Ribosylhomocysteinase
- Author
-
Jinge Zhu, Gang Shen, Charles E. Bell, Dehua Pei, and Rakhi Rajan
- Subjects
Models, Molecular ,animal structures ,Stereochemistry ,Stereoisomerism ,Crystallography, X-Ray ,Structure-Activity Relationship ,chemistry.chemical_compound ,Bacterial Proteins ,mental disorders ,Drug Discovery ,Ribose ,Structure–activity relationship ,Binding site ,Caproates ,Homocysteine ,chemistry.chemical_classification ,Binding Sites ,Hydroxamic acid ,biology ,food and beverages ,Active site ,Dioxolanes ,Lyase ,Amides ,Anti-Bacterial Agents ,Butyrates ,Carbon-Sulfur Lyases ,Enzyme ,chemistry ,Drug Design ,biology.protein ,bacteria ,Molecular Medicine ,Carbamates ,sense organs ,Bacillus subtilis - Abstract
S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2. Inhibitors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of antibacterial agents. LuxS utilizes a divalent metal ion as a Lewis acid during catalysis. In this work, a series of structural analogues of the substrate SRH and a 2-ketone intermediate were designed and synthesized. Kinetic studies indicate that the compounds act as reversible, competitive inhibitors against LuxS, with the most potent inhibitors having K(I) values in the submicromolar range. These represent the most potent LuxS inhibitors that have been reported to date. Cocrystal structures of LuxS bound with two of the inhibitors largely confirmed the design principles, i.e., the importance of both the homocysteine and ribose moieties in high-affinity binding to the LuxS active site.
- Published
- 2006
26. Crystal Structure of S-Ribosylhomocysteinase (LuxS) in Complex with a Catalytic 2-Ketone Intermediate
- Author
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Xubo Hu, Jinge Zhu, Rakhi Rajan, Charles E. Bell, and Dehua Pei
- Subjects
Models, Molecular ,Ketone ,Hydrolases ,Stereochemistry ,Crystal structure ,Arginine ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Substrate Specificity ,chemistry.chemical_compound ,Bacterial Proteins ,Thioether ,Ribose ,Hydrolase ,Serine ,Histidine ,Cysteine ,Ferrous Compounds ,Homocysteine ,Vibrio ,chemistry.chemical_classification ,Alanine ,Binding Sites ,Cobalt ,Ketones ,Carbon-Sulfur Lyases ,chemistry ,Catalytic cycle ,Mutagenesis, Site-Directed ,Spectrophotometry, Ultraviolet ,Crystallization ,Isomerization ,Bacillus subtilis - Abstract
S-Ribosylhomocysteinase (LuxS) is an Fe(2+)-dependent metalloenzyme that catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial quorum-sensing molecule. The proposed mechanism involves an initial metal-catalyzed aldose-ketose isomerization reaction, which results in the migration of the ribose carbonyl group from its C1 to C2 position and the formation of a 2-ketone intermediate. A repetition of the isomerization reaction shifts the carbonyl group to the C3 position. Subsequent beta-elimination reaction at the C4 and C5 positions completes the catalytic cycle. In this work, a catalytically inactive mutant (C84A) of Co(2+)-substituted Bacillus subtilis LuxS was cocrystallized with the 2-ketone intermediate and the structure was determined to 1.8 A resolution. The structure reveals that the C2 carbonyl oxygen is directly coordinated with the metal ion, providing strong support for the proposed Lewis acid function of the metal ion during catalysis. Cys-84 and Glu-57 are optimally positioned to act as general acids/bases during the isomerization and elimination reactions. In addition, Ser-6, His-11, and Arg-39 are involved in substrate/ intermediate binding through hydrogen bonding interactions. The above conclusions are further confirmed by site-directed mutagenesis and visible absorption spectroscopic studies.
- Published
- 2005
27. Biochemical characterization of the topoisomerase domain of Methanopyrus kandleri topoisomerase V
- Author
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Amy K. Osterman, Rakhi Rajan, Alexandra T. Gast, and Alfonso Mondragón
- Subjects
Archaeal Proteins ,Static Electricity ,Euryarchaeota ,DNA and Chromosomes ,medicine.disease_cause ,Biochemistry ,Catalysis ,Mass Spectrometry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Catalytic Domain ,medicine ,Amino Acids ,Molecular Biology ,chemistry.chemical_classification ,Mutation ,biology ,Topoisomerase ,Circular Dichroism ,Mutagenesis ,Active site ,Cell Biology ,DNA ,Hydrogen-Ion Concentration ,Amino acid ,Enzyme ,chemistry ,DNA Topoisomerases, Type I ,biology.protein ,Mutagenesis, Site-Directed ,DNA supercoil - Abstract
Topoisomerases are ubiquitous enzymes that modify the topological state of DNA inside the cell and are essential for several cellular processes. Topoisomerase V is the sole member of the type IC topoisomerase subtype. The topoisomerase domain has a unique fold among topoisomerases, and the putative active site residues show a distinct arrangement. The present study was aimed at identifying the roles of the putative active site residues in the DNA cleavage/religation process. Residues Arg-131, Arg-144, His-200, Glu-215, Lys-218, and Tyr-226 were mutated individually to a series of conservative and non-conservative amino acids, and the DNA relaxation activity at different pH values, times, and enzyme concentrations was compared with wild-type activity. The results suggest that Arg-144 is essential for protein stability because any substitution at this position was deleterious and that Arg-131 and His-200 are involved in transition state stabilization. Glu-215 reduces the DNA binding ability of topoisomerase V, especially in shorter fragments with fewer helix-hairpin-helix DNA binding motifs. Finally, Lys-218 appears to play a direct role in catalysis but not in charge stabilization of the protein-DNA intermediate complex. The results suggest that although catalytically important residues are oriented in different fashions in the active sites of type IB and type IC topoisomerases, similar amino acids play equivalent roles in both of these subtypes of enzymes, showing convergent evolution of the catalytic mechanism.
- Published
- 2014
28. Topoisomerases
- Author
-
Rakhi Rajan, Chandra Critchelow, and Alfonso Mondragón
- Published
- 2013
29. Probing the Catalytic Mechanism of S-Ribosylhomocysteinase (LuxS) with Catalytic Intermediates and Substrate Analogues
- Author
-
Bhaskar Gopishetty, Rakhi Rajan, Dehua Pei, Charles E. Bell, Jinge Zhu, Stanislaw F. Wnuk, and Adam J. Sobczak
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Time Factors ,Stereochemistry ,Kinetics ,Cleavage (embryo) ,Biochemistry ,Catalysis ,Article ,Substrate Specificity ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Thioether ,Bacterial Proteins ,Catalytic Domain ,Ribose ,Organic chemistry ,Molecule ,Homocysteine ,chemistry.chemical_classification ,Molecular Structure ,Substrate (chemistry) ,General Chemistry ,Ketones ,Carbon-Sulfur Lyases ,Enzyme ,chemistry ,Biocatalysis - Abstract
S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a beta-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.
- Published
- 2009
30. Probing the DNA sequence specificity of Escherichia coli RECA protein
- Author
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Rakhi Rajan, James W. Wisler, and Charles E. Bell
- Subjects
Oligonucleotides ,Repressor ,DNA, Single-Stranded ,Biology ,medicine.disease_cause ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Viral Proteins ,Adenosine Triphosphate ,0103 physical sciences ,Genetics ,medicine ,Escherichia coli ,Nucleotide ,Viral Regulatory and Accessory Proteins ,Binding site ,030304 developmental biology ,Repetitive Sequences, Nucleic Acid ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,010304 chemical physics ,Oligonucleotide ,Escherichia coli Proteins ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,DNA-Binding Proteins ,Repressor Proteins ,Rec A Recombinases ,chemistry ,Biochemistry ,Nucleic acid ,bacteria ,Homologous recombination ,DNA ,Protein Binding - Abstract
Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5'-GCTGGTGG-3'). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a lambda repressor fragment in the presence of RecA, ADP-AlF4 and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.
- Published
- 2006
31. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis
- Author
-
Rakhi Rajan, Noelle Wakefield, and Erik J. Sontheimer
- Subjects
Biophysics ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,Endonuclease ,Bacterial Proteins ,Structural Biology ,Staphylococcus epidermidis ,Genetics ,RNA Precursors ,CRISPR ,CRISPR-associated ,Cas6 ,Clustered regularly interspaced short palindromic repeats ,Molecular Biology ,030304 developmental biology ,Trans-activating crRNA ,0303 health sciences ,CRISPR interference ,biology ,Base Sequence ,Models, Genetic ,030306 microbiology ,RNA ,Cell Biology ,biology.organism_classification ,Endonucleases ,3. Good health ,RNA processing ,biology.protein ,Nucleic acid ,Nucleic Acid Conformation ,Electrophoresis, Polyacrylamide Gel ,CRISPR-Cas Systems ,Biogenesis - Abstract
In many bacteria and archaea, an adaptive immune system (CRISPR–Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR–Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR–Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3′ half of the CRISPR repeat for recognition and processing.
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