Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.

Department of Analysis of Biologically Important Molecular Complexes Masaryk University 612 65 Brno Czech Republic

Odvolání publikace

Mol Cell Biol. 2024;44(6):259. doi: 10.1080/10985549.2024.2358694. PubMed

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Aihara H, Ito Y, Kurumizaka H, Yokoyama S, Shibata T. The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR. J Mol Biol. 1999;290:495–504. PubMed

Ausubel F M, Brent R, Kingston R, Morre D, Seidman J, Smith A, Struhl K. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1994.

Bai Y, Symington L S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 1996;10:2025–2037. PubMed

Bendixen C, Gangloff S, Rothstein R. A yeast mating-selection scheme for detection of protein-protein interactions. Nucleic Acids Res. 1994;22:1778–1779. PubMed PMC

Boeke J D, LaCroute F, Fink G R. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197:345–346. PubMed

Chien C T, Bartel P L, Sternglanz R, Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci USA. 1991;88:9578–9582. PubMed PMC

Clever B, Interthal H, Schmuckli-Maurer J, King J, Sigrist M, Heyer W D. Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins. EMBO J. 1997;16:2535–2544. PubMed PMC

Cuff J A, Clamp M E, Siddiqui A S, Finlay M, Barton G J. JPred: a consensus secondary structure prediction server. Bioinformatics. 1998;14:892–893. PubMed

Donovan J W, Milne G T, Weaver D T. Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair. Genes Dev. 1994;8:2552–2562. PubMed

Game J C. Radiation-sensitive mutants and repair in yeast. In: Spencer J F T, Spencer D, Smith A R W, editors. Yeast genetics: fundamental and applied aspects. New York, N.Y: Springer-Verlag; 1983. pp. 105–137.

Gasior S L, Wong A K, Kora Y, Shinohara A, Bishop D K. Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes. Genes Dev. 1998;12:2208–2221. PubMed PMC

Gietz R D, Schiestl R H, Willems A R, Woods R A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast. 1995;11:355–360. PubMed

Golub E I, Kovalenko O V, Gupta R C, Ward D C, Radding C M. Interaction of human recombination proteins Rad51 and Rad54. Nucleic Acids Res. 1997;25:4106–4110. PubMed PMC

Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. PubMed

Hays S L, Firmenich A A, Berg P. Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc Natl Acad Sci USA. 1995;92:6925–6929. PubMed PMC

James P, Halladay J, Craig E A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 1996;144:1425–1436. PubMed PMC

Jiang H, Xie Y, Houston P, Stemke-Hale K, Mortensen U H, Rothstein R, Kodadek T. Direct association between the yeast Rad51 and Rad54 recombination proteins. J Biol Chem. 1996;271:33181–33186. PubMed

Johnson R D, Symington L S. Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol Cell Biol. 1995;15:4843–4850. PubMed PMC

Klein H L. RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis. Genetics. 1997;147:1533–1543. PubMed PMC

Konola J T, Nastri H G, Logan K M, Knight K L. Mutations at Pro67 in the RecA protein P-loop motif differentially modify coprotease function and separate coprotease from recombination activities. J Biol Chem. 1995;270:8411–8419. PubMed

Kurumizaka H, Aihara H, Kagawa W, Shibata T, Yokoyama S. Human Rad51 amino acid residues required for Rad52 binding. J Mol Biol. 1999;291:537–548. PubMed

Lambert S, Lopez B S. Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms. EMBO J. 2000;19:3090–3099. PubMed PMC

Laskowski R A, McArthur M W, Moss D S, Thornton J M. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr. 1993;26:283–291.

Lim D S, Hasty P. A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol. 1996;16:7133–7143. PubMed PMC

Luthy R, Bowie J U, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992;356:83–85. PubMed

Marmorstein L Y, Ouchi T, Aaronson S A. The BRCA2 gene product functionally interacts with p53 and RAD51. Proc Natl Acad Sci USA. 1998;95:13869–13874. PubMed PMC

Mazin A V, Bornarth C J, Solinger J A, Heyer W, Kowalczykowski S C. Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament. Mol Cell. 2000;6:1–20. PubMed

Mazin A V, Zaitseva E, Sung P, Kowalczykowski S C. Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing. EMBO J. 2000;19:1148–1156. PubMed PMC

Menetski J P, Kowalczykowski S C. Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J Mol Biol. 1985;181:281–295. PubMed

Menetski J P, Varghese A, Kowalczykowski S C. Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein. Biochemistry. 1988;27:1205–1212. PubMed

Milne G T, Weaver D T. Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52. Genes Dev. 1993;7:1755–1765. PubMed

Mortensen U H, Bendixen C, Sunjevaric I, Rothstein R. DNA strand annealing is promoted by the yeast Rad52 protein. Proc Natl Acad Sci USA. 1996;93:10729–10734. PubMed PMC

New J H, Sugiyama T, Zaitseva E, Kowalczykowski S C. Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature. 1998;391:407–410. PubMed

Ogawa T, Yu X, Shinohara A, Egelman E H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science. 1993;259:1896–1899. PubMed

Palmer B R, Marinus M G. DNA methylation alters the pattern of spontaneous mutation in Escherichia coli cells (mutD) defective in DNA polymerase III proofreading. Mutat Res. 1991;264:15–23. PubMed

Petes T D, Malone R E, Symington L S. Recombination in yeast. In: Broach J R, Pringle J R, Jones E W, editors. The molecular and cellular biology of the yeast Saccharomyces: genome dynamics, protein synthesis, and energetics. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1991. pp. 407–521.

Petukhova G, Van Komen S, Vergano S, Klein H, Sung P. Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. J Biol Chem. 1999;274:29453–29462. PubMed

Roca A I, Cox M M. RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol. 1997;56:129–223. PubMed

Sali A, Blundell T L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234:779–815. PubMed

Saraste M, Sibbald P R, Wittinghofer A. The P-loop–a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990;15:430–434. PubMed

Schild D. Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. Genetics. 1995;140:115–127. PubMed PMC

Shen Z, Cloud K G, Chen D J, Park M S. Specific interactions between the human RAD51 and RAD52 proteins. J Biol Chem. 1996;271:148–152. PubMed

Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992;69:457–470. PubMed

Shinohara A, Ogawa T. Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature. 1998;391:404–407. PubMed

Shinohara M, Shita-Yamaguchi E, Buerstedde J M, Shinagawa H, Ogawa H, Shinohara A. Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis. Genetics. 1997;147:1545–1556. PubMed PMC

Sippl M J. Recognition of errors in three-dimensional structures of proteins. Proteins. 1993;17:355–362. PubMed

Story R M, Weber I T, Steitz T A. The structure of the E. coli recA protein monomer and polymer. Nature. 1992;355:318–325. PubMed

Sturzbecher H W, Donzelmann B, Henning W, Knippschild U, Buchhop S. p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J. 1996;15:1992–2002. PubMed PMC

Sugawara N, Ivanov E L, Fishman-Lobell J, Ray B L, Wu X, Haber J E. DNA structure-dependent requirements for yeast RAD genes in gene conversion. Nature. 1995;373:84–86. PubMed

Sugiyama T, New J H, Kowalczykowski S C. DNA annealing by Rad52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc Natl Acad Sci USA. 1998;95:6049–6054. PubMed PMC

Sugiyama T, Zaitseva E M, Kowalczykowski S C. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J Biol Chem. 1997;272:7940–7945. PubMed

Sung P. Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J Biol Chem. 1997;272:28194–28197. PubMed

Sung P. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 1997;11:1111–1121. PubMed

Sung P, Robberson D L. DNA strand exchange mediated by a Rad51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell. 1995;82:453–461. PubMed

Thomas B J, Rothstein R. The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics. 1989;123:725–738. PubMed PMC

Van Dyck E, Stasiak A Z, Stasiak A, West S C. Binding of double-strand breaks in DNA by human Rad52 protein. Nature. 1999;398:728–731. PubMed

Whitehouse I, Flaus A, Cairns B R, White M F, Workman J L, Owen-Hughes T. Nucleosome mobilization catalysed by the yeast SWI/SNF complex. Nature. 1999;400:784–787. PubMed

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