Elastic Behavior of RecA-DNA Helical Filaments

Nishinaka, Taro; Doi, Yuko; Hara, Reiko; Yashima, Eiji

doi:10.1016/j.jmb.2007.05.044

Cited by 16 publications

(19 citation statements)

References 37 publications

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“…This conversion is a direct response of the RecA–ssDNA filament to ATP concentration and is well explained by Michaelis–Menten kinetics (Figure 5a). Direct conversion between extended and compressed filament states reported here and previously (51), is at odds with studies that found a stoichiometry of 5 nt instead of 3 nt per monomer for the compressed ADP-bound filament (29). We expect that an active filament bound to nucleotide triplets maintains its stoichiometry upon ATP hydrolysis as adjacent RecA-bound nucleotides are unavailable.…”

Section: Discussioncontrasting

confidence: 99%

Dynamics of RecA filaments on single-stranded DNA

Loenhout

Heijden

Kanaar

et al. 2009

Nucleic Acids Research

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RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA–ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA–ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.

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Section: Discussioncontrasting

confidence: 99%

Dynamics of RecA filaments on single-stranded DNA

Loenhout

Heijden

Kanaar

et al. 2009

Nucleic Acids Research

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“…Although the ADP-bound state has low DNA affinity, Rad51 does not necessarily dissociate from ssDNA upon ATP hydrolysis. Dissociation of RAD51-ADP is concentrated at filament ends (38), whereas internal monomers, stabilized by interaction with two adjacent monomers, exchange ADP back for ATP without dissociating (39,40). This general behavior has also been observed with bacterial RecA protein, which hydrolyzes ATP ϳ100 times faster than eukaryotic Rad51 ( Fig.…”

mentioning

confidence: 71%

“…2A). In single molecule experiments, switching between a buffer with and without ATP, RecA filaments elongate and shorten, respectively (40). The local changes in filament pitch that occur with ATP hydrolysis promote nucleoprotein filament movement (41).…”

mentioning

confidence: 99%

Homologous recombination and the repair of DNA double-strand breaks

Wright

Shah

Heyer

2018

Journal of Biological Chemistry

506

450

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Homologous recombination enables the cell to access and copy intact DNA sequence information in , particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes. Aspects of DNA topology affecting individual steps are highlighted. Overall, recombination is a dynamic pathway with multiple metastable and reversible intermediates designed to achieve DNA repair with high fidelity.

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“…These structural transitions take place without an intervening step requiring dissociation of Rad51 into monomers. Importantly, a recent single molecule study has demonstrated that bacterial RecA bound to ssDNA can also undergo repeated cycles of elongation and compression (24), and another study found that human Rad51 can transition from an extended to a compressed filament (19). Thus, reversible structural transitions may be a common attribute of this family of DNA recombinases.…”

Section: Discussionmentioning

confidence: 99%

Structural transitions within human Rad51 nucleoprotein filaments

Robertson¹,

Moses

Kwon

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.DNA curtain ͉ homologous recombination ͉ single molecule imaging D ouble-stranded DNA breaks (DSBs) are 1 of the most deleterious forms of DNA damage and can lead to cell death or oncogenic transformation. Homologous recombination (HR) is an evolutionarily conserved pathway used to repair DSBs, and is essential for maintaining genomic stability (1, 2). When a DSB occurs, the 5Ј ends of the DNA are resected, yielding long 3Ј single-stranded DNA (ssDNA) overhangs, which are the loading site for a DNA recombinase. The recombinase aligns the ssDNA with a homologous double stranded DNA (dsDNA) and then invades the duplex to form a D-loop. The invading end can then serve as a primer for the replication machinery, which uses the homologous duplex as a template, and the resulting products are resolved to restore the continuity of the chromosomes.The DNA transactions that take place during HR are mediated by members of the RAD52 epistasis group of proteins (1-3). This includes the recombinase Rad51, which plays a central role in HR and assembles into a nucleoprotein filament on the ssDNA overhangs generated at the DSB (4, 5). This filament is responsible for catalyzing the pairing, alignment, and strand invasion steps during recombination (6). Rad51 is sufficient to catalyze these reactions in vitro (7), however, numerous accessory factors are required in vivo and their functions range from facilitating Rad51 loading at the outset of the reaction to promoting the disassembly of Rad51 upon completion of strand invasion (1,3,8,9).Human Rad51 has a flexible N-terminal domain and a central ATP-binding cor...

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Elastic Behavior of RecA-DNA Helical Filaments

Cited by 16 publications

References 37 publications

Dynamics of RecA filaments on single-stranded DNA

Dynamics of RecA filaments on single-stranded DNA

Homologous recombination and the repair of DNA double-strand breaks

Structural transitions within human Rad51 nucleoprotein filaments

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