ABC  Vol.3 No.3 , June 2013
Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in S. cerevisiae

Several modes of eukaryotic of DNA double strand break repair (DSBR) depend on synapsis of complementary DNA. The Rad51 ATPase, the S. cerevisiae homolog of E. coli RecA, plays a key role in this process by catalyzing homology searching and strand exchange between an invading DNA strand and a repair template (e.g. sister chromatid or homologous chromosome). Synthesis dependent strand annealing (SDSA), a mode of DSBR, requires Rad51. Another repair enzyme, the Rad1-Rad10 endonuclease, acts in the final stages of SDSA, hydrolyzing 3 overhanging single-stranded DNA. Here we show in vivo by fluo-rescence microscopy that the ATP binding function of yeast Rad51 is required to recruit Rad10 SDSA sites indicating that Rad51 pre-synaptic filament formation must occur prior to the recruitment of Rad1-Rad10. Our data also show that Rad51 ATPase activity, an important step in Rad51 filament disassembly, is not absolutely required in order to recruit Rad1- Rad10 to DSB sites.

Cite this paper
Karlin, J. and Fischhaber, P. (2013) Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in S. cerevisiae. Advances in Biological Chemistry, 3, 295-303. doi: 10.4236/abc.2013.33033.
[1]   Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R.A., et al. (2005) DNA repair and mutagenesis. 2nd Edition, ASM Press, Washington DC.

[2]   Paques, F. and Haber, J.E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 63, 349404.

[3]   Symington, L.S. (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiology and Molecular Biology Reviews, 66, 630-670. doi:10.1128/MMBR.66.4.630-670.2002

[4]   New, J.H., Sugiyama, T., Zaitseva, E. and Kowalczykowski, S.C. (1998) Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature, 391, 407-410. doi:10.1038/34950

[5]   Sugiyama, T., New, J.H. and Kowalczykowski, S.C. (1998) DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proceedings of the National Academy of Sciences of the United States of America, 95, 6049-6054. doi:10.1073/pnas.95.11.6049

[6]   Sugiyama, T., Zaitseva, E.M. and Kowalczykowski, S.C. (1997) A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. The Journal of Biological Chemistry, 272, 7940-7945. doi:10.1074/jbc.272.12.7940

[7]   Sung, P. (1997) Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. The Journal of Biological Chemistry, 272, 28194-28197. doi:10.1074/jbc.272.45.28194

[8]   Maloisel, L., Fabre, F. and Gangloff, S. (2008) DNA polymerase delta is preferentially recruited during homologous recombination to promote heteroduplex DNA extension. Molecular and Cellular Biology, 28, 1373-1382. doi:10.1128/MCB.01651-07

[9]   Sugiyama, T., Kantake, N., Wu, Y. and Kowalczykowski, S.C. (2006) Rad52-mediated DNA annealing after Rad51mediated DNA strand exchange promotes second ssDNA capture. The EMBO Journal, 25, 5539-5548. doi:10.1038/sj.emboj.7601412

[10]   Bardwell, A.J., Bardwell, L., Johnson, D.K. and Friedberg, E.C. (1993) Yeast DNA recombination and repair proteins Rad1 and Rad10 constitute a complex in vivo mediated by localized hydrophobic domains. Molecular Microbiology, 8, 1177-1188. doi:10.1111/j.1365-2958.1993.tb01662.x

[11]   Fishman-Lobell, J. and Haber, J.E. (1992) Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science, 258, 480-484. doi:10.1126/science.1411547

[12]   Sugawara, N., Ira, G. and Haber, J.E. (2000) DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. Molecular and Cellular Biology, 20, 5300-5309. doi:10.1128/MCB.20.14.5300-5309.2000

[13]   Sugawara, N., Paques, F., Colaiacovo, M. and Haber, J.E. (1997) Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination. Proceedings of the National Academy of Sciences of the United States of America, 94, 9214-9219. doi:10.1073/pnas.94.17.9214

[14]   Moore, D.M., Karlin, J., Gonzalez-Barrera, S., Mardiros, A., Lisby, M., et al. (2009) Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae. Nucleic Acids Research, 37, 6429-6438.

[15]   Antony, E., Tomko, E.J., Xiao, Q., Krejci, L., Lohman, T.M., et al. (2009) Srs2 disassembles Rad51 filaments by a protein-protein interaction triggering ATP turnover and dissociation of Rad51 from DNA. Molecular Cell, 35, 105-115. doi:10.1016/j.molcel.2009.05.026

[16]   Sung, P. and Stratton, S.A. (1996) Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis. The Journal of Biological Chemistry, 271, 27983-27986. doi:10.1074/jbc.271.45.27983

[17]   Lisby, M., Rothstein, R. and Mortensen, U.H. (2001) Rad52 forms DNA repair and recombination centers during S phase. Proceedings of the National Academy of Sciences of the United States of America, 98, 8276-8282. doi:10.1073/pnas.121006298

[18]   Lisby, M., Mortensen, U.H. and Rothstein, R. (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nature Cell Biology, 5, 572577. doi:10.1038/ncb997

[19]   Barlow, J.H., Lisby, M. and Rothstein, R. (2008) Differential regulation of the cellular response to DNA double-strand breaks in G1. Molecular Cell, 30, 73-85. doi:10.1016/j.molcel.2008.01.016

[20]   Thomas, B.J. and Rothstein, R. (1989) Elevated recombination rates in transcriptionally active DNA. Cell, 56, 619-630. doi:10.1016/0092-8674(89)90584-9

[21]   Zhao, X., Muller, E.G. and Rothstein, R. (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Molecular Cell, 2, 329-340. doi:10.1016/S1097-2765(00)80277-4

[22]   Morgan, E.A., Shah, N. and Symington, L.S. (2002) The requirement for ATP hydrolysis by Saccharomyces cerevisiae Rad51 is bypassed by mating-type heterozygosity or RAD54 in high copy. Molecular and Cellular Biology, 22, 6336-6343. doi:10.1128/MCB.22.18.6336-6343.2002

[23]   Mardiros, A., Benoun, J.M., Haughton, R., Baxter, K., Kelson, E. P., et al. (2011) Rad10-YFP focus induction in response to UV depends on RAD14 in yeast. Acta Histochemica, 113, 409-415. doi:10.1016/j.acthis.2010.03.005

[24]   Lisby, M., Barlow, J.H., Burgess, R.C. and Rothstein, R. (2004) Choreography of the DNA damage response: Spatiotemporal relationships among checkpoint and repair proteins. Cell, 118, 699-713. doi:10.1016/j.cell.2004.08.015

[25]   Chi, P., Van Komen, S., Sehorn, M.G., Sigurdsson, S. and Sung, P. (2006) Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amsterdam), 5, 381-391. doi:10.1016/j.dnarep.2005.11.005

[26]   Li, F., Dong, J., Pan, X., Oum, J.H., Boeke, J.D., et al. (2008) Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates. Molecular Cell, 30, 325335. doi:10.1016/j.molcel.2008.02.028

[27]   Mazon, G., Lam, A.F., Ho, C.K., Kupiec, M. and Symington, L.S. (2012) The Rad1-Rad10 nuclease promotes chromosome translocations between dispersed repeats. Nature Structural & Molecular Biology, 19, 964-971. doi:10.1038/nsmb.2359

[28]   Fung, C.W., Fortin, G.S., Peterson, S.E. and Symington, L.S. (2006) The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation. Molecular and Cellular Biology, 26, 9544-9554. doi:10.1128/MCB.00599-06

[29]   Pfander, B., Moldovan, G.L., Sacher, M., Hoege, C. and Jentsch, S. (2005) SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature, 436, 428-433. doi:10.1038/nature03665

[30]   Rockmill, B., Fung, J.C., Branda, S.S. and Roeder, G.S. (2003) The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over. Current Biology, 13, 19541962. doi:10.1016/j.cub.2003.10.059

[31]   Watt, P.M., Louis, E.J., Borts, R.H. and Hickson, I.D. (1995) Sgs1: A eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation. Cell, 81, 253-260. doi:10.1016/0092-8674(95)90335-6

[32]   Yeung, M. and Durocher, D. (2011) Srs2 enables checkpoint recovery by promoting disassembly of DNA damage foci from chromatin. DNA Repair (Amsterdam), 10, 1213-1222. doi:10.1016/j.dnarep.2011.09.005

[33]   Bugreev, D.V., Yu, X., Egelman, E.H. and Mazin, A.V. (2007) Novel pro- and anti-recombination activities of the Bloom’s syndrome helicase. Genes & Development, 21, 3085-3094. doi:10.1101/gad.1609007

[34]   Ira, G., Malkova, A., Liberi, G., Foiani, M. and Haber, J. E. (2003) Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell, 115, 401-411. doi:10.1016/S0092-8674(03)00886-9