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Rad54 dissociates homologous recombination intermediates by branch migration

Abstract

Double-strand DNA breaks (DSBs) cause cell death and genome instability. Homologous recombination is a major DSB repair pathway that operates by forming joint molecules with homologous DNA sequences, which are used as templates to achieve accurate repair. In eukaryotes, Rad51 protein (RecA homolog) searches for homologous sequences and catalyzes the formation of joint molecules (D-loops). Once joint molecules have been formed, DNA polymerase extends the 3′ single-stranded DNA tails of the broken chromosome, restoring the lost information. How joint molecules subsequently dissociate is unknown. We reconstituted DSB repair in vitro using purified human homologous recombination proteins and DNA polymerase η. We found that Rad54 protein, owing to its ATP-dependent branch-migration activity, can cause dissociation of joint molecules. These results suggest a previously uncharacterized mechanism of DSB repair in which Rad54 branch-migration activity plays an important role.

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Figure 1: Scheme illustrating the alternative pathways of homologous recombination.
Figure 2: RAD54 promotes dissociation of joint molecules (D-loops).
Figure 3: RAD54 promotes dissociation of synthetic D-loops by branch migration.
Figure 4: In vitro reconstitution of DSB repair.
Figure 5: RAD52 promotes formation of double D-loops in the presence of RPA.
Figure 6: RAD54 promotes dissociation of double D-loops produced by RAD51 and RAD52 proteins.
Figure 7: RAD54 promotes dissociation of linear double D-loops through branch migration of Holliday junctions.

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Acknowledgements

We thank P. Sung (Yale University), M. Wold (University of Iowa) and E. Golub (Yale University) for RAD51, RPA and RAD52 expression vectors; G. Schnitzler and N. Ulyanova (Tufts University) for human SWI/SNF protein; Z. Zhang and R. Kingston (Harvard Medical School) for RAD54B protein; and M. Bouchard, M. Rossi and O. Mazina (Drexel University College of Medicine) for comments and discussion. This work was supported by US National Institutes of Health grant CA100839 to A.V.M.

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Correspondence to Alexander V Mazin.

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Bugreev, D., Hanaoka, F. & Mazin, A. Rad54 dissociates homologous recombination intermediates by branch migration. Nat Struct Mol Biol 14, 746–753 (2007). https://doi.org/10.1038/nsmb1268

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