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.     

Recombination and the Progression of Meiosis in Saccharomyces Cerevisiae

Recombination is an essential part of meiosis; in almost all organisms, including Saccharomyces cerevisiae, proper chromosome segregation and the viability of meiotic products is dependent upon normal levels of recombination. In this article we examine the kinetics of the meiotic divisions in four mutants defective in the initiation of recombination. We find that mutations in any of three Early Exchange genes (REC104, REC114 or REC102) confer a phenotype in which the reductional division occurs earlier than in an isogenic wild-type diploid. We also present data confirming previous reports that strains with a mutation in the Early Exchange gene MEI4 undergo the first division at about the same time as wild-type cells. The rec104 mutation is epistatic to the mei4 mutation for the timing of the first division. These observations suggest a possible relationship between the initiation of recombination and the timing of the reductional division. These data also allow these four Early Exchange genes examined to be distinguished in terms of their role in coordinating recombination with the reductional division.     

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.     

Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.     

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.     

Mitotic Recombination among Subtelomeric Y' Repeats in Saccharomyces Cerevisiae

Y's are a dispersed family of repeats that vary in copy number, location and restriction fragment lengths between strains but exhibit within-strain homogeneity. We have studied mitotic recombination between members of the subtelomeric Y' repeated sequence family of Saccharomyces cerevisiae. Individual copies of Y's were marked with SUP11 and URA3 which allowed for the selection of duplications and losses of the marked Y's. Duplications occurred by ectopic recombinational interactions between Y's at different chromosome ends as well as by unequal sister chromatid exchange. Several of the ectopic duplications resulted in an originally Y'-less chromosome end acquiring a marked Y'. Among losses, most resulted from ectopic exchange or conversion in which only the marker sequence was lost. In some losses, the chromosome end became Y'-less. Although the two subsets of Y's, Y'-longs (6.7 kb) and Y'-shorts (5.2 kb), share extensive sequence homology, a marked Y' recombines highly preferentially within its own subset. These mitotic interactions can in part explain the maintenance of Y's and their subsets, the homogeneity among Y's within a strain, as well as diversity between strains.     

Requirement of Mismatch Repair Genes Msh2 and Msh3 in the Rad1-Rad10 Pathway of Mitotic Recombination in Saccharomyces Cerevisiae

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3Δ mutation has an effect on recombination similar to that of the rad1Δ and rad10Δ mutations. The msh2Δ mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, 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.     

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.     

The PCNA Interaction Protein Box Sequence in Rad54 Is an Integral Part of Its ATPase Domain and Is Required for Efficient DNA Repair and Recombination

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.     

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 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.     

Rad9, Rad17, and Rad24 Are Required for S Phase Regulation in Saccharomyces Cerevisiae in Response to DNA Damage

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9Δ, rad17Δ, and rad24Δ. Furthermore, the triple rad9Δ rad17Δ rad24Δ mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1Δ, mag1Δ, apn1Δ, rev3Δ, rad18Δ, rad16Δ, dun1-Δ100, sad4-1, tel1Δ, rad26Δ, rad51Δ, rad52-1, rad54Δ, rad14Δ, rad1Δ, pol30-46, pol30-52, mad3Δ, pds1Δ/esp2Δ, pms1Δ, mlh1Δ, and msh2Δ are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.     

Cis-Acting Determinants Affecting Centromere Function, Sister-Chromatid Cohesion and Reciprocal Recombination during Meiosis in Saccharomyces Cerevisiae

We have employed a system that utilizes homologous pairs of human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to assess the specific role (s) of conserved centromere DNA elements (CDEI, CDEII and CDEIII) in meiotic chromosome disjunction fidelity. Thirteen different centromere (CEN) mutations were tested for their effects on meiotic centromere function. YACs containing a wild-type CEN DNA sequence segregate with high fidelity in meiosis I (99% normal segregation) and in meiosis II (96% normal segregation). YACs containing a 31-bp deletion mutation in centromere DNA element II (CDEIIδ31) in either a heterocentric (mutant/wild type), homocentric (mutant/mutant) or monosomic (mutant/--) YAC pair configuration exhibited high levels (16-28%) of precocious sister-chromatid segregation (PSS) and increased levels (1-6%) of nondisjunction meiosis I (NDI). YACs containing this mutation also exhibit high levels (21%) of meiosis II nondisjunction. Interestingly, significant alterations in homolog recombination frequency were observed in the exceptional PSS class of tetrads, suggesting unusual interactions between prematurely separated sister chromatids and their homologous nonsister chromatids. We also have assessed the meiotic segregation effects of rare gene conversion events occurring at sites located immediately adjacent to or distantly from the centromere region. Proximal gene conversion events were associated with extremely high levels (60%) of meiosis I segregation errors (including both PSS and NDI), whereas distal events had no apparent effect. Taken together, our results indicate a critical role for CDEII in meiosis and underscore the importance of maintaining sister-chromatid cohesion for proper recombination in meiotic prophase and for proper disjunction in meiosis I.     

Rad9 Is Required for B Cell Proliferation and Immunoglobulin Class Switch Recombination

B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9^−/− B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9^−/− mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9^−/− B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.     

Saccharomyces Cerevisiae Rad52 Alleles Temperature-Sensitive for the Repair of DNA Double-Strand Breaks

We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to γ-radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, interchromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third.     

Genetic and Physical Analysis of Double-Strand Break Repair and Recombination in Saccharomyces Cerevisiae

We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.     

Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.     

The Saccharomyces Cerevisiae Ku Autoantigen Homologue Affects Radiosensitivity Only in the Absence of Homologous Recombination

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G(1) and G(2) or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.