Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors

Peter Chi 2 YoungHo Kwon 2 Mari-Liis Visnapuu 1 Isabel Lam 0 Sergio R. Santa Maria 0 Xiuzhong Zheng 0 Anastasiya Epshtein 0 Eric C. Greene 1 3 Patrick Sung 2 Hannah L. Klein 0 0 Department of Biochemistry and NYU Cancer Institute, New York University School of Medicine , 550 First Avenue, New York, NY 10016 1 Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center 2 Molecular Biophysics and Biochemistry, Yale University School of Medicine , 333 Cedar Street, New Haven, CT 06520 3 Howard Hughes Medical Institute , 650 West 168th Street, New York, NY 10032 USA The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair. - Rad54 and Rdh54 are members of the Swi2 protein family. These evolutionarily conserved proteins possess dsDNA-dependent ATPase activity that fuels their translocation on dsDNA, resulting in DNA supercoiling and transient strand unwinding. Both proteins physically interact with the recombinase Rad51 and synergize with the Rad51ssDNA nucleoprotein filament to promote D-loop formation, DNA branch migration and chromatin remodeling, all of which are essential steps in homologous recombination (HR) (1). Interestingly, Rad54 and Rdh54 both can remove Rad51 from dsDNA in vitro. The ability to dissociate the Rad51dsDNA complex has been postulated to be important for releasing Rad51 from bulk chromatin, to ensure that a sufficient pool of free recombinase is available for repair and to prevent the accumulation of toxic Rad51DNA intermediates. Moreover, removal of Rad51 by Rad54 and Rdh54 may be necessary to allow access of a DNA polymerase to the primer terminus in the newly made D-loop during HR. RAD54 and RDH54 likely serve distinct functions in mitotic and meiotic recombination, as mutants have distinct phenotypes (24). rad54 mutants are sensitive to DNA damaging agents and have significant reduction in mitotic recombination whereas rdh54D are only slightly sensitive to DNA damage and have a modest reduction in interchromosomal recombination, but are not affected in intrachromosomal recombination. rdh54D diploids have The authors wish it to be known that, in their opinion, the first four authors should be regarded as joint First Authors. significant meiotic recombination defects and are delayed in the repair of meiotic double strand breaks whereas rad54D diploids does not show a delay in the repair of meiotic double strand breaks, although spore viability is reduced. Even though both Rad54 and Rdh54 can dissociate Rad51 from dsDNA in vitro (5,6), whether these proteins remove Rad51 from chromatin in vivo and the functional significance and relative contributions of Rad54 and Rdh54 toward Rad51 clearance from chromatin remain unanswered. In vivo, RAD54 has a more important role than RDH54 in the recombinational repair of methyl methanesulfonate (MMS) damaged DNA and gaps that occur from replication across a damaged DNA template, as seen by the strong MMS sensitivity of rad54 mutants, while rdh54 mutants show only a modest sensitivity (3,4). In a recent study we have found that Rdh54 has a critical role in removing Rad51 from chromatin when Rad51 is expressed in excess, while Rad54 has only a minor role in this regard (7). In contrast, following irradiation damage, Rad51 foci persist in rad54 mutants, but not in rdh54 mutants (7). Likewise, we do not yet know whether Uls1, another Swi2 family member that was originally identified based on its two-hybrid interaction with the meiotic recombinase Dmc1, also plays a role in Rad51 clearance from chromatin (8). ULS1 has no known role in DNA repair, as deletion of the gene does not render cells sensitive to DNA damaging agents, although it appears to play a role in removing excess Rad51 from chromatin (7). Here, we show synthetic growth deficiency and DNA damage sensitivity of double and triple mutants of the aforementioned Swi2-like factors that can be efficiently suppressed by deleting RAD51. In congruence with this, a genomic suppressor of the rad54D uls1D mutant defects is shown to harbor a mutation, A265V, in RAD51. We provide evidence that the suppressor activity of rad51A265V stems from the combined effect of this mutation on the affinity of Rad51 for DNA and the accelerated removal of the mutant rad51 protein by Rdh54 from dsDNA. Taken together, our results provide compelling evidence for a cytotoxic effect of gratuitous Rad51dsDNA complexes and suggest that Rad54, Rdh54 and Uls1 all contribute toward clearance of these toxic nucleoprotein complexes in a hierarchical fashion. Additionally, our results suggest that Rad54 and Rdh54 recognize different Rad51dsDNA complexes in vivo. MATERIALS AND METHODS Spot assays on MMS-supplemented plates Yeast cultures were incubated overnight at 30 C in YPD medium. After determining cell density, the cultures were adjusted to 107 cells/ml and then serially diluted. Aliquots of 4 ml from the serial dilutions were spotted onto SC or SC-containing MMS at the indicated concentration. SC plates containing MMS were made directly before use. The plates were then incubated at 30 C for 56 days. Screen for suppressors of rad54 uls1 MMS sensitivity The rad54D uls1D strain used for EMS mutagenesis is HKY1287-11B MATa rad54::LEU2 uls1::KANMX leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 hom3-10 RAD5. For EMS mutagenesis, cells were grown overnight at 30 C, collected and washed twice with water and resuspended in an equal volume of 0.1 M sodium phosphate buffer (pH 7). The exact cell density was determined with a hemacytometer and adjusted to 2 108 cells/ml. Two 1-ml aliquots were made, and 0.5 ml EMS (Sigma) was added to one aliquot, while the other aliquot served as control. The tubes were vortexed vigorously before incubating for 1 h at 30 C with agitation. After incubation, the cells were collected and washed three times with 8 ml 5% sodiu (...truncated)