Requirement of Mismatch Repair Genes MSH 2 and MSH 3 in the RLu ) I - RADIU Pathway of Mitotic Recombination in Saccharomyces cerevisiae

The RADl and RAD10 genes of Saccharomyces cereuisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Radl-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH? function in mitotic recombination. For both his? and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh?A mutation has an effect on recombination similar to that of the r u d l A and rad l0A mutations. The msh2A mutation also reduces the rate of recombination of the his? duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH? function in the RAD1-RADIO recombination pathway, and studies presented here suggest an involvement of the RADl-RADIO pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Radl, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process. I N both prokaryotes and eukaryotes, nucleotide excision repair (NER) represents an important mechanism that removes DNA damage inflicted by UV light, bulky chemical agents, and DNA crosslinking agents. In humans, a defect in NER causes xeroderma pigmentosum (XP) . Cell fusion studies have thus far identified seven complementation groups, XP-A to XP-G, and all of these mutant XP cell lines exhibit a deficiency in the incision step of excision repair of UV damaged DNA. XP cells are hypermutable by UV light, and as a consequence, XP patients suffer from a high incidence of skin cancers (reviewed in CLEAVER and KRAEMER 1989). Additional human NER genes (ERCCs) have been identified by cross complementation of the excision defect of UV sensitive rodent cell lines. The complexity of NER in the yeast Saccharomyces cereuisiae resembles that in humans. Initial genetic studies identified 11 genes to have a role in this process in yeast. Mutations in seven of these genes, RADl, RAD2, RAD3, RAD4, RADIO, RAD14, and RAD25, render cells highly sensitive to UV light and defective in the incision step of NER. The structure and function of NER genes have been conserved to a remarkable degree among eukaryotes from yeast to humans (for a review, see P a KASH et ul. 1993; FRIEDBERG et ul. 1995). The evolutionary conservation of the NER genes supports the premise that information obtained from the yeast system can be utilized for deciphering the role of these genes in NER and in other cellular processes in humans. Cowaponding author: Satya Prakash, Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Building, 11th and Mechanic St., Galveston, TX 77555-1061. Genetics 1 4 2 727-756 (March, 1996) Genetic studies with the yeast NER genes have revealed that some of them are multifunctional, acting in processes other than excision repair. Specifically, RAD3 and RAD25 are essential for cell viability because of their requirement in RNA polymerase I1 transcription (QIU et al. 1993; GUZDER et al. 1994a,b). The Rad3 and Rad25 proteins comprise two of the subunits of Pol I1 transcription factor IIH (TFIIH) (FEAVER et al. 1993; SVEJSTRUP et al. 1994). The RADl and RAD10 genes function in mitotic recombination; these genes are required for intrachromosomal recombination between direct repeats and they also affect the homologous integration of DNA molecules into genomic sequences (SCHIESTL and PRAKASH 1988, 1990). Epistasis analyses have indicated that RADl and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 double strand break recombination pathway (SCHIESTL and PRAKASH 1988, 1990). The effects of RADl on mitotic recombination of direct and inverted repeats have been analyzed genetically in many other studies and mutations in R A D l also lower recombination stimulated by RNA polymerase I or RNA polymerase I1 transcription (KLEIN 1988; ACUILERA and KLEIN 1989; THOMAS and ROTHSTEIN 1989; ZEHFUS et al. 1990; BAILIS et al. 1992; LIEFSHITZ et al. 1995; R A ~ R A Y and SYMINGTON 1995). The RADl and RAD10 genes also have been shown to have a role in the repair of a double strand break, induced by the action of HO endonuclease, via a single strand annealing (SSA) pathway (FISHMAN-LOBELL and HABER 1992; IVANOV and HABER 1995), where repair of a double strand break is accomplised by deletion of one of the direct repeats and loss of the intervening sequence. 728 M . Saparbaev, I>. Prakash and s. Prakash The Radl and Rad10 proteins are associated in a tight complex i n vivo. Studies with a rad1 mutation that inactivates complex formation with Rad10 have suggested that complex formation underlies the biological action of these proteins (BAILLY et nl. 1992). The RadlRad10 complex has a DNA endonuclease activity, and it exhibits a structure specific nuclease activity that cleaves 3’-ended single stranded DNA at its junction with the duplex region of DNA (SUNG e1 al. 1993; TOMKINSON et al. 1993; BAKDWELL et al. 1994). With the aim of identifying other genes that function in the Radl-Rad10 recombination pathway, we have examined the effects of mismatch repair (MMR) genes MSH2, MSH3, PMS1, and M L H l on mitotic recombination. Five MSH genes, MSHl, MSH2, MSH3, MSH4, and MSH5, have so far been identified in S . cerevisiae. The MSHl encoded protein functions in mitochondria, and the MSH4 and MSH5 genes have a role in reciprocal exchange (crossing over) between homologs in meiosis (ROSS-MACDONALD and ROEIER 1994; HOLLINGSWOKTH et al. 1995). The MSH2 and MSH3 genes participate in DNA mismatch repair; null mutations in these genes result in elevated rates of spontaneous mutations and in an increase in the rate of postmeiotic segregation that results from the lack of repair of heteroduplex DNA generated during meiotic recombination, and they also cause an increase in instability of simple repetitive DNA sequences (REENAN and KOLODNER 1992; NEW et al. 1993; STRAND el nl. 1993). The effect of the msh3 null mutation on mismatch repair, however, is less marked than that of the msh2 null mutation (REENAN and KOLOIINER 1992; NEW et al. 1993). Purified Msh2 protein binds specifically to DNA containing mismatched base pairs and insertions (FISMEI. et nl. 1994; AI.ANI et al. 1995). Mutations i n PMSl and M L H l also cause a defect in mismatch repair and they result in marked increases in spontaneous mutation rates, in postmeiotic segregation, and in instability of simple sequence DNA repeats (STRAND et al. 1993; PROLLA et al. 1994). Recently, null mutations in MSH2 and PMS2, the latter being the mammalian counterpart of yeast PMSl, have been generated in mice. These mutations cause defects in MMR, including microsatellite instability, and mutations in PMS2 but not in MSH2 also cause male sterility (BAKER et nl. 1995; DE MrIND et nl. 1995). Here, we show that the MSH2 and MSH3 genes function in mitotic recombination, and epistasis analyses indicate the involvement of these genes in the M l M l O recombination pathway. However, we find no evidence for a role of PMSl and MLHl in mitotic recombination. We discuss the possible role of coupling of mismatch binding proteins with the recombinational machinery in preventing recombination between related but diverged (homeologous) sequences that occur at many sites in the eukaryotic genome. MATERIAIS AND METHODS Strains and media: Eschrriclain coli DH5a [ F mdA1 hsdR17 .rupE44 [hi-I recAI gyrA96 relAl A(lrrU169480 dlacA AM15)l was used for plasmid propagation. Yeast strains RSY6 ( M 7 a urn3-52 lPu2-3, -1 12 trp5-27 urg4-3 adr2-40 ih1-92 HIS?::pR%) and LP2752-4B (MAT0 lysl-1 ura3-52 his4-864,-1176 pBR313 his4-260, -39) were from our laboratory stocks and have been described (S(:HIESIL. and PKUASII 1988). YPD, minimal, and synthetic complete media for growth of yeast strains were as described (SHERMAN rt a1. 1986). Transformation and other procedures: Yeast strains were transformed using a lithium acetate method (ITO et al. 1983; S(:HIESTI. and GIETZ 1989). Plasmid purification from E. roli and electrophoresis of DNA were performed as described (MmI,vns rt al. 1982). DNA fragments were isolated from agarose gels using the SpinBind DNA Extraction Unit (FMS BioProducts, Rockland, ME) as recommended by the manufacturer. Deletion mutations of RAD and mismatch repair genes: Yeast strains RSY6 and L2P2752-4B were deleted for the R A D 1 and RAD10 genes (radA) by replacing the entire or most of the open reading frame of each gene with the S. rrruuisiae URA3 gene flanked by Salmonrlla typhirnuriurn his(; sequences (AIANI rt al. 1987) by the gene replacement method (ROTHSTEIN 1991). The genomic RAD1 and RAD10 genes were replaced in strain RSY6 and in strain LP2752-4B with plasmids pR1.6 and pR10.25, respectively, resulting in isogenic m d l A and mdl0A derivatives. Plasmid pR1.6, a derivative ofpUC19, contains an EcoRI-SaZl DNA insert of the following composition: starting at the EcoRI site of pUC19, there is a 480-bp DNA fragment of 5’ R.iD1 region DNA that extends to position +40 of the RALll open reading frame (OW), where + I is the A of the initiating ATG (RWNOI.I)S rl nl. 1987). This is followed by the 3.8-kb BnrnHI fragment, from plasmid pDC82. This DNA fragment contains the yeast URA? gene flanked by the duplicated S. typhinawiuna hisGgene ( A I A N I rt al. 1987). which is referred to as the “gene blaster” fragment. The gene blaster BnrnHI fragment was derived by inserting the BamHI-BglII hi.rC>URA3-hi.\(; fragment from pNKY51 (AIANI rt rcl. 1987) into the XOaI site of pUC18 following Klenow treatment to blunt the ends. This generates BarnHI sites at either end, thus enabling removal of this fragment from pDG82 by digestion with BamHI. The gene blaster fragment is followed by 650 bp of 3’ region RAD1 DNA, which begins at position +3211 of the 3300-bp RAI>l OW. The Hind111 site at the 3’ end of this 650-bp fragment has been converted to a S n l I site. Thus, generation of a genomic mdlA mutation is achieved by transformation of urn? yeast cells to URA? with the EcoRI-SalI fragment ofpR1.6. This results in a mdlA mutant that is lacking the KAL)l sequence from positions +41 to +3210. Plasmid pR10.25, used to generate radl0A mutations, was constructed in an essentially similar manner except that pUC8 was used instead of pUC19. In pR10.25, the 3.8-kb BmnHI gene blaster fragment replaces nucleotides +50 to +473 of the 630 nucleotide long RAD10 ORF (REYNo~.~)~ rl nl. 1985). Generation of a radlOA mutation is achieved by transformation of ura? yeast to LIRA? with EcoRI-Sall digested pR10.25. The M I 5 2 gene was disrupted with URA? by transformation of RSY6 and LP2752-4B with RamHI-digested plasmid pSM22, obtained from D. S(:HlI.T). Plasmid pMS22 i s a PBR322 derivative containing the RAD52 gene disrupted with L’RA?. A genomic d 5 2 A mutation w a s made by digestion of pMS22 with BalnHI and transformation with the BankHI fragment in which the URA? gene had been inserted at the BgAI site in the RAD52 OW. The deletion mutation of each RAD gene