Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

2003 ◽  
Vol 36 (4) ◽  
pp. 429-453 ◽  
Author(s):  
Chantal Prévost ◽  
Masayuki Takahashi
Keyword(s):  
Homologous Recombination ◽  
Structural Information ◽  
Double Helix ◽  
Genetic Material ◽  
Strand Exchange ◽  
Double Stranded Dna ◽  
Dna Deformation ◽  
Architectural Proteins ◽  
Dna Strands ◽  
Dna Strand

1. Introduction 4302. Transformations of the RecA filament 4312.1 The different forms of the RecA filament 4312.2 Orientation and position of the RecA monomers in the active filament 4332.3 Transmission of structural information along the filament 4333. RecA-induced DNA deformations 4353.1 Characteristics of RecA-bound DNA 4353.2 Stretching properties of double-stranded DNA 4363.3 DNA bound to architectural proteins 4373.4 Implications for RecA-induced DNA deformations 4383.5 Axial distribution of the DNA stretching deformation 4384. Contacts between RecA and the DNA strands 4404.1 The DNA-binding sites 4404.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 4425. Strand arrangement during pairing reorganization 4445.1 Hypotheses for DNA strand association 4445.2 Association via major or minor grooves 4465.3 Post-strand exchange geometries 4466. Conclusion 4477. Acknowledgments 4488. References 448Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

scholarly journals Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination

2019 ◽  
Vol 47 (15) ◽  
pp. 7798-7808
Author(s):  
Benjamin Boyer ◽  
Claudia Danilowicz ◽  
Mara Prentiss ◽  
Chantal Prévost
Keyword(s):  
Homologous Recombination ◽  
Atp Hydrolysis ◽  
Strand Exchange ◽  
Geometrical Approach ◽  
Structural Level ◽  
Dna Double Strand Breaks ◽  
Sequence Recognition ◽  
Dna Strands ◽  
Reverse Strand ◽  
Dna Strand

Abstract Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.

scholarly journals The homologous recombination machinery modulates the formation of RNA–DNA hybrids and associated chromosome instability

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Lamia Wahba ◽  
Steven K Gore ◽  
Douglas Koshland
Keyword(s):  
Homologous Recombination ◽  
Genome Instability ◽  
Chromosome Instability ◽  
Rna Degradation ◽  
Strand Exchange ◽  
Hybrid Formation ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Yeast Mutants

Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo.

scholarly journals Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

2018 ◽  
Author(s):  
Daan C. Swarts ◽  
Martin Jinek
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Cleavage Product ◽  
List Type ◽  
Francisella Novicida ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Dna Strands ◽  
Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

scholarly journals Homologous Recombination in Real Time: DNA Strand Exchange by RecA

2008 ◽  
Vol 30 (4) ◽  
pp. 530-538 ◽  
Author(s):  
Thijn van der Heijden ◽  
Mauro Modesti ◽  
Susanne Hage ◽  
Roland Kanaar ◽  
Claire Wyman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Real Time ◽  
Strand Exchange ◽  
Dna Strand Exchange ◽  
Dna Strand

scholarly journals BRCA2 regulates DMC1-mediated recombination through the BRC repeats

2016 ◽  
Vol 113 (13) ◽  
pp. 3515-3520 ◽  
Author(s):  
Juan S. Martinez ◽  
Catharina von Nicolai ◽  
Taeho Kim ◽  
Åsa Ehlén ◽  
Alexander V. Mazin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Spatial Organization ◽  
Strand Exchange ◽  
Molecule Formation ◽  
Brca2 Protein ◽  
Dna Strand Exchange ◽  
Functional Consequences ◽  
Strand Invasion ◽  
Dna Strand ◽  
Stimulation Of

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1–3 and 6–8. However, BRC1–3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6–8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1–ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA–ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.

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