Abstract
We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations--one a deletion of amino acids 210-327 and the other a missense mutation of residue 235--increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.
Full Text
The Full Text of this article is available as a PDF (197.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adzuma K., Ogawa T., Ogawa H. Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2735–2744. doi: 10.1128/mcb.4.12.2735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Asleson E. N., Okagaki R. J., Livingston D. M. A core activity associated with the N terminus of the yeast RAD52 protein is revealed by RAD51 overexpression suppression of C-terminal rad52 truncation alleles. Genetics. 1999 Oct;153(2):681–692. doi: 10.1093/genetics/153.2.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Awasthi S., Palmer R., Castro M., Mobarak C. D., Ruby S. W. New roles for the Snp1 and Exo84 proteins in yeast pre-mRNA splicing. J Biol Chem. 2001 Jun 25;276(33):31004–31015. doi: 10.1074/jbc.M100022200. [DOI] [PubMed] [Google Scholar]
- Bai Y., Symington L. S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 1996 Aug 15;10(16):2025–2037. doi: 10.1101/gad.10.16.2025. [DOI] [PubMed] [Google Scholar]
- Boundy-Mills K. L., Livingston D. M. A Saccharomyces cerevisiae RAD52 allele expressing a C-terminal truncation protein: activities and intragenic complementation of missense mutations. Genetics. 1993 Jan;133(1):39–49. doi: 10.1093/genetics/133.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brill S. J., Sternglanz R. Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell. 1988 Jul 29;54(3):403–411. doi: 10.1016/0092-8674(88)90203-6. [DOI] [PubMed] [Google Scholar]
- Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
- Davis A. P., Symington L. S. The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing. Genetics. 2001 Oct;159(2):515–525. doi: 10.1093/genetics/159.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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 Nov 1;8(21):2552–2562. doi: 10.1101/gad.8.21.2552. [DOI] [PubMed] [Google Scholar]
- Dornfeld K. J., Livingston D. M. Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Apr;11(4):2013–2017. doi: 10.1128/mcb.11.4.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Firmenich A. A., Elias-Arnanz M., Berg P. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52. Mol Cell Biol. 1995 Mar;15(3):1620–1631. doi: 10.1128/mcb.15.3.1620. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ghislain M., Udvardy A., Mann C. S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase. Nature. 1993 Nov 25;366(6453):358–362. doi: 10.1038/366358a0. [DOI] [PubMed] [Google Scholar]
- Guo W., Grant A., Novick P. Exo84p is an exocyst protein essential for secretion. J Biol Chem. 1999 Aug 13;274(33):23558–23564. doi: 10.1074/jbc.274.33.23558. [DOI] [PubMed] [Google Scholar]
- 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 U S A. 1995 Jul 18;92(15):6925–6929. doi: 10.1073/pnas.92.15.6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hays S. L., Firmenich A. A., Massey P., Banerjee R., Berg P. Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA. Mol Cell Biol. 1998 Jul;18(7):4400–4406. doi: 10.1128/mcb.18.7.4400. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heinemeyer W., Gruhler A., Möhrle V., Mahé Y., Wolf D. H. PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins. J Biol Chem. 1993 Mar 5;268(7):5115–5120. [PubMed] [Google Scholar]
- Hilt W., Enenkel C., Gruhler A., Singer T., Wolf D. H. The PRE4 gene codes for a subunit of the yeast proteasome necessary for peptidylglutamyl-peptide-hydrolyzing activity. Mutations link the proteasome to stress- and ubiquitin-dependent proteolysis. J Biol Chem. 1993 Feb 15;268(5):3479–3486. [PubMed] [Google Scholar]
- Ivanov E. L., Sugawara N., Fishman-Lobell J., Haber J. E. Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae. Genetics. 1996 Mar;142(3):693–704. doi: 10.1093/genetics/142.3.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson E. S., Blobel G. Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. J Biol Chem. 1997 Oct 24;272(43):26799–26802. doi: 10.1074/jbc.272.43.26799. [DOI] [PubMed] [Google Scholar]
- Johnson R. D., Symington L. S. Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol Cell Biol. 1995 Sep;15(9):4843–4850. doi: 10.1128/mcb.15.9.4843. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Katz W. S., Solomon F. Diversity among beta-tubulins: a carboxy-terminal domain of yeast beta-tubulin is not essential in vivo. Mol Cell Biol. 1988 Jul;8(7):2730–2736. doi: 10.1128/mcb.8.7.2730. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaytor M. D., Livingston D. M. Allele-specific suppression of temperature-sensitive mutations of the Saccharomyces cerevisiae RAD52 gene. Curr Genet. 1996 Feb;29(3):203–210. doi: 10.1007/BF02221549. [DOI] [PubMed] [Google Scholar]
- Kaytor M. D., Livingston D. M. Saccharomyces cerevisiae RAD52 alleles temperature-sensitive for the repair of DNA double-strand breaks. Genetics. 1994 Aug;137(4):933–944. doi: 10.1093/genetics/137.4.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lisby M., Rothstein R., Mortensen U. H. Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8276–8282. doi: 10.1073/pnas.121006298. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Malkova A., Ivanov E. L., Haber J. E. Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7131–7136. doi: 10.1073/pnas.93.14.7131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milne G. T., Ho T., Weaver D. T. Modulation of Saccharomyces cerevisiae DNA double-strand break repair by SRS2 and RAD51. Genetics. 1995 Mar;139(3):1189–1199. doi: 10.1093/genetics/139.3.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milne G. T., Weaver D. T. Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52. Genes Dev. 1993 Sep;7(9):1755–1765. doi: 10.1101/gad.7.9.1755. [DOI] [PubMed] [Google Scholar]
- Mortensen U. H., Bendixen C., Sunjevaric I., Rothstein R. DNA strand annealing is promoted by the yeast Rad52 protein. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10729–10734. doi: 10.1073/pnas.93.20.10729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nguyen M. M., Livingston D. M. Cold-sensitive rad52 alleles of yeast. Curr Genet. 1997 Aug;32(2):100–107. doi: 10.1007/s002940050253. [DOI] [PubMed] [Google Scholar]
- Nguyen M. M., Livingston D. M. The effect of a suppressed rad52 mutation on the suppression of rad6 by srs2. Yeast. 1997 Sep 15;13(11):1059–1064. doi: 10.1002/(SICI)1097-0061(19970915)13:11<1059::AID-YEA165>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
- Rattray A. J., Symington L. S. Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination. Genetics. 1994 Nov;138(3):587–595. doi: 10.1093/genetics/138.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Santocanale C., Neecke H., Longhese M. P., Lucchini G., Plevani P. Mutations in the gene encoding the 34 kDa subunit of yeast replication protein A cause defective S phase progression. J Mol Biol. 1995 Dec 8;254(4):595–607. doi: 10.1006/jmbi.1995.0641. [DOI] [PubMed] [Google Scholar]
- 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 May;140(1):115–127. doi: 10.1093/genetics/140.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwarz S. E., Matuschewski K., Liakopoulos D., Scheffner M., Jentsch S. The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme. Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):560–564. doi: 10.1073/pnas.95.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shen Z., Pardington-Purtymun P. E., Comeaux J. C., Moyzis R. K., Chen D. J. Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system. Genomics. 1996 Oct 15;37(2):183–186. doi: 10.1006/geno.1996.0540. [DOI] [PubMed] [Google Scholar]
- Shen Z., Pardington-Purtymun P. E., Comeaux J. C., Moyzis R. K., Chen D. J. UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins. Genomics. 1996 Sep 1;36(2):271–279. doi: 10.1006/geno.1996.0462. [DOI] [PubMed] [Google Scholar]
- Shinohara A., Ogawa T. Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature. 1998 Jan 22;391(6665):404–407. doi: 10.1038/34943. [DOI] [PubMed] [Google Scholar]
- Signon L., Malkova A., Naylor M. L., Klein H., Haber J. E. Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break. Mol Cell Biol. 2001 Mar;21(6):2048–2056. doi: 10.1128/MCB.21.6.2048-2056.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Song B., Sung P. Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange. J Biol Chem. 2000 May 26;275(21):15895–15904. doi: 10.1074/jbc.M910244199. [DOI] [PubMed] [Google Scholar]
- Umezu K., Sugawara N., Chen C., Haber J. E., Kolodner R. D. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism. Genetics. 1998 Mar;148(3):989–1005. doi: 10.1093/genetics/148.3.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van Velzen-Mol H. W., Blankespoor M. N., Wagenaar-Fischer M. M., van Leerdam F. J. De standaard 'Opsporing visuele stoornissen 0-19 jaar' van de jeugdgezondheidszorg. Ned Tijdschr Geneeskd. 2003 Oct 11;147(41):2012–2017. [PubMed] [Google Scholar]