Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.     

Different Types of Recombination Events Are Controlled by the Rad1 and Rad52 Genes of Saccharomyces Cerevisiae

Intrachromosomal recombination within heteroallelic duplications located on chromosomes III and XV of Saccharomyces cerevisiae has been examined. Both possible orientations of alleles have been used in each duplication. Three recombinant classes, gene conversions, pop-outs and triplications, were recovered. Some of the recombinant classes were not anticipated from the particular allele orientation of the duplication. Recovery of these unexpected recombinants requires the RAD1 gene. These studies show that RAD1 has a role in recombination between repeated sequences, and that the recombination event is a gene conversion associated with a crossover. These events appear to involve very localized conversion of a heteroduplex region and are distinct from RAD52 mediated gene conversion events. Evidence is also presented to suggest that most recombination events between direct repeats are intrachromatid, not between sister chromatids.     

A Defect in Mismatch Repair in Saccharomyces Cerevisiae Stimulates Ectopic Recombination between Homeologous Genes by an Excision Repair Dependent Process

Null mutations in three recombination and DNA repair genes were studied to determine their effects on mitotic recombination between the duplicate AdoMet (S-adenosylmethionine) synthetase genes (SAM1 and SAM2) in Saccharomyces cerevisiae. SAM1 and SAM2, located on chromosomes XII and IV, respectively, encode functionally equivalent although differentially regulated AdoMet synthetases. These similar but not identical (homeologous) genes are 83% homologous at the nucleotide level and this identity is limited solely to the coding regions of the genes. Single frameshift mutations were introduced into the 5' end of SAM1 and the 3' end of SAM2 by restriction site ablation. The sequences surrounding these mutations differ significantly in their degree of homology to the corresponding area of the other gene. Mitotic ectopic recombination between the mutant sam genes occurs at a rate of 8.4 x 10(-9) in a wild-type genetic background. Gene conversion of the marker within the region of greater sequence homology occurs 20-fold more frequently than conversion of the marker within the region of relative sequence diversity. The relative orientation of the two genes prevents the recovery of translocations. Mitotic recombination between the sam genes is completely dependent on the DNA repair and recombination gene RAD52. A mutation in PMS1, a mismatch repair gene, causes a 4.5-fold increase in the rate of ectopic recombination. RAD1, an excision repair gene, is required to observe this increased rate of ectopic conversion. In addition, RAD1 is involved in modulating the pattern of coconversion during recombination between the homeologous sam genes. These results suggest that interactions between mismatch repair, excision repair and recombinational repair functions are involved in determining the ectopic gene conversion frequency between the sam genes.     

Distance-Independence of Mitotic Intrachromosomal Recombination in Saccharomyces Cerevisiae

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.     

Use of a Chromosomal Inverted Repeat to Demonstrate That the Rad51 and Rad52 Genes of Saccharomyces Cerevisiae Have Different Roles in Mitotic Recombination

An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 X 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 had functions in addition to those of the Rad51/Rad52 protein complex.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

Interaction between Ustilago Maydis Rec2 and Rad51 Genes in DNA Repair and Mitotic Recombination

A gene encoding a Ustilago maydis Rad51 orthologue has been isolated. rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and γ radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.     

Rdh54, a Rad54 Homologue in Saccharomyces Cerevisiae, Is Required for Mitotic Diploid-Specific Recombination and Repair and for Meiosis

Most mitotic recombination and repair genes of Saccharomyces cerevisiae show no specificity of action for the genome ploidy. We describe here a novel repair and recombination gene that is specific for recombination and repair between homologous chromosomes. The RDH54 gene is homologous to the RAD54 gene, but rdh54 mutants do not show sensitivity to methyl methanesulfonate at concentrations that sensitize a rad54 mutant. However, the rdh54 null mutation enhances the methyl methanesulfonate sensitivity of a rad54 mutant and single rdh54 mutants are sensitive to prolonged exposure at high concentrations of methyl methanesulfonate. The RDH54 gene is required for recombination, but only in a diploid. We present evidence showing that the RDH54 gene is required for interhomologue gene conversion but not intrachromosomal gene conversion. The rdh54 mutation confers diploid-specific lethalities and reduced growth in various mutant backgrounds. These phenotypes are due to attempted recombination. The RDH54 gene is also required for meiosis as homozygous mutant diploids show very poor sporulation and reduced spore viability. The role of the RDH54 gene in mitotic repair and in meiosis and the pathway in which it acts are discussed.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

Characterization of Insertion Mutations in the Saccharomyces Cerevisiae Msh1 and Msh2 Genes: Evidence for Separate Mitochondrial and Nuclear Functions

The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (< 0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (< 0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus.     

Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.     

Effects of Mutations of Rad50, Rad51, Rad52, and Related Genes on Illegitimate Recombination in Saccharomyces Cerevisiae

To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rad51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.     

Position Effects in Ectopic and Allelic Mitotic Recombination in Saccharomyces Cerevisiae

We have examined the role that genomic location plays in mitotic intragenic recombination. Mutant alleles of the LEU2 gene were inserted at five locations in the yeast genome. Diploid and haploid strains containing various combinations of these inserts were used to examine both allelic recombination (between sequences at the same position on parental homologs) and ectopic recombination (between sequences at nonallelic locations). Chromosomal location had little effect on mitotic allelic recombination. The rate of recombination to LEU2 at five different loci varied less than threefold. This finding contrasts with previous observations of strong position effects in meiosis; frequencies of meiotic recombination at the same five loci differ by about a factor of forty. Mitotic recombination between dispersed copies of leu2 displayed strong position effects. Copies of leu2 located approximately 20 kb apart on the same chromosome recombined at rates 6-13-fold higher than those observed for allelic copies of leu2. leu2 sequences located on nonhomologous chromosomes or at distant loci on the same chromosome recombined at rates similar to those observed for allelic copies. We suggest that, during mitosis, parental homologs interact with each other no more frequently than do nonhomologous chromosomes.     

Saccharomyces cerevisiae Msh2p and Msh6p ATPase Activities Are Both Required during Mismatch Repair

In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that were predicted to disrupt ATP binding, ATP hydrolysis, or both activities in each subunit were created. Mutations in either subunit resulted in a mismatch repair defect, and overexpression of either mutant subunit in a wild-type strain resulted in a dominant negative phenotype. Msh2p-Msh6p complexes bearing one or both mutant subunits were analyzed for binding to DNA containing base pair mismatches. None of the mutant complexes displayed a significant defect in mismatch binding; however, unlike wild-type protein, all mutant combinations continued to display mismatch binding specificity in the presence of ATP and did not display ATP-dependent conformational changes as measured by limited trypsin protease digestion. Both wild-type complex and complexes defective in the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh6p ATPase, however, displayed weak ATPase activities that were unaffected by the presence of DNA substrate. The results from these studies suggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex play important and coordinated roles in postmismatch recognition steps that involve ATP hydrolysis. Furthermore, our data support a model whereby Msh6p uses its ATP binding or hydrolysis activity to coordinate mismatch binding with additional mismatch repair components.     

The Srs2 Suppressor of Rad6 Mutations of Saccharomyces Cerevisiae Acts by Channeling DNA Lesions into the Rad52 DNA Repair Pathway

rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.     

Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.