Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

Characterization of Mutations That Suppress the Temperature-Sensitive Growth of the Hpr1Δ Mutant of Saccharomyces Cerevisiae

The hpr1Δ3 mutant of Saccharomyces cerevisiae is temperature-sensitive for growth at 37° and has a 1000-fold increase in deletion of tandem direct repeats. The hyperrecombination phenotype, measured by deletion of a leu2 direct repeat, is partially dependent on the RAD1 and RAD52 gene products, but mutations in these RAD genes do not suppress the temperature-sensitive growth phenotype. Extragenic suppressors of the temperature-sensitive growth have been isolated and characterized. The 14 soh (suppressor of hpr1) mutants recovered represent eight complementation groups, with both dominant and recessive soh alleles. Some of the soh mutants suppress hpr1 hyperrecombination and are distinct from the rad mutants that suppress hpr1 hyperrecombination. Comparisons between the SOH genes and the RAD genes are presented as well as the requirement of RAD genes for the Soh phenotypes. Double soh mutants have been analyzed and reveal three classes of interactions: epistatic suppression of hpr1 hyperrecombination, synergistic suppression of hpr1 hyperrecombination and synthetic lethality. The SOH1 gene has been cloned and sequenced. The null allele is 10-fold increased for recombination as measured by deletion of a leu2 direct repeat.     

HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene.

The HPR1 gene has been cloned by complementation of the hyperrecombination phenotype of hpr1-1 strains by using a color assay system. HPR1 is a gene that is in single copy on chromosome IV of Saccharomyces cerevisiae, closely linked to ARO1, and it codes for a putative protein of 752 amino acids (molecular mass, 88 kilodaltons). Computer searches revealed homology (48.8% conserved homology; 24.8% identity) with the S. cerevisiae TOP1 gene in an alpha-helical stretch of 129 amino acids near the carboxy-terminal region of both proteins. The ethyl methanesulfonate-induced hpr1-1 mutation is a single-base change that produces a stop codon at amino acid 559 coding for a protein that lacks the carboxy-terminal TOP1 homologous region. Haploid strains carrying deletions of the HPR1 gene show a slightly reduced mitotic growth rate and extremely high rates of intrachromosomal excision recombination (frequency, 10 to 15%) but have a undetectable effect on rDNA recombination. Double-null mutants hpr1 top1 grow very poorly. We conclude that Hpr1 is a novel eucaryotic protein, mutation of which causes an increase in mitotic intrachromosomal excision recombination, and that it may be functionally related to an activity of the topoisomerase I protein.     

The Hyper-Gene Conversion Hpr5-1 Mutation of Saccharomyces Cerevisiae Is an Allele of the Srs2/Radh Gene

The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion. This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway. The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase. The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X. The hpr5-1 allele contains missense mutation in the putative ATP binding domain. A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms. We propose that the HPR5 gene functions in the RAD6 repair pathway.     

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.     

Isolation and Genetic Analysis of Extragenic Suppressors of the Hyper-Deletion Phenotype of the Saccharomyces Cerevisiae Hpr1δ Mutation

The HPR1 gene of Saccharomyces cerevisae is involved in maintaining low levels of deletions between DNA repeats. To understand how deletions initiate in the absence of the Hpr1 protein and the mechanisms of recombination leading to deletions in S. cerevisiae, we have isolated mutations as suppressors of the hyper-deletion phenotype of the hpr1δ mutation. The mutations defined five different genes called HRS for hyper-recombination suppression. They suppress the hyper-deletion phenotype of hpr1δ strains for three direct repeat systems tested. The mutations eliminated the hyper-deletion phenotype of hpr1δ strains either completely (hrs1-1 and hrs2-1) or significantly (hrs3-1, hrs4-1 and hrs5-1). None of the mutations has a clear effect on the levels of spontaneous and double-strand break-induced deletions. Among other characteristics we have found are the following: (1) one mutation, hrs1-1, reduces the frequency of deletions in rad52-1 strains 20-fold, suggesting that the HRS1 gene is involved in the formation of RAD52-independent deletions; (2) the hrs2-1 hpr1δ mutant is sensitive to methyl-methane-sulfonate and the single mutants hpr1δ and hrs2-1 are resistant, which suggests that the HPR1 and HRS2 proteins may have redundant DNA repair functions; (3) the hrs4-1 mutation confers a hyper-mutator phenotype and (4) the phenotype of lack of activation of gene expression observed in hpr1δ strains is only partially suppressed by the hrs2-1 mutation, which suggests that the possible functions of the Hpr1 protein in gene expression and recombination repair can be separated. We discuss the possible relationship between the HPR1 and the HRS genes and their involvement in initiation of the events responsible for deletion formation.     

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.     

Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1Δ strains

Null hprl Δ strains show a large increase (up to 2000-fold) over wild type in the frequency of occurrence of deletions between direct repeats on three different chromosomes. However, we show that hprl Δ mutations have little or no effect on reciprocal exchange, gene conversion or unequal sister chromatid exchange, as determined using intrachromosomal, interchromosomal and plasmid-chromosome assay systems. A novel intrachromosomal recombination system has allowed us to determine that over 95% of deletions in hpr1 Δ strains do not occur by reciprocal exchange. On the other hand, hpr1 Δ strains show chromosome loss frequencies of up to 100 times the wild-type level. Our results suggest that yeast cells have a very efficient non-conservative recombination mechanism, dependent on RADI and RAD52, that causes deletions between direct DNA repeats, and this mechanism is strongly stimulated in hpr1 Δ strains. The results indicate that the Hpr1 protein is required for stability of DNA repeats and chromosomes. We propose that in the absence of the Hprl protein the cell destabilizes the genome by allowing the initiation of events that lead to deletions of sequences between repeats, and to chromosome instability. We discuss the roles that proteins such as Hprl have in maintaining direct repeats and in preventing non-conservative recombination and consider the importance of these functions for chromosome stability.     

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.     

RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes.     

Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Recombination in Aspergillus

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.     

Spontaneous Mitotic Recombination in Yeast: The Hyper-Recombinational Rem1 Mutations Are Alleles of the Rad3 Gene

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.     

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

The RAD1 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RAD1 gene on genetic recombination. Mitotic intrachromosomal and interchromosomal recombination in RAD+, rad1, rad52, and other rad mutant strains was examined. The rad1 deletion mutation and some rad1 point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the rad1 rad52 double mutant, indicating that the RAD1 and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RAD1 gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the rad1 delta mutation did lower the frequency of integration of linear plasmids and DNA fragments into homologous genomic sequences. We suggest that RAD1 plays a role in recombination after the formation of the recombinogenic substrate.     

Isolation and Characterization of Schizosaccharomyces Pombe Mutants Affected in Mitotic Recombination

A haploid Schizosaccharomyces pombe strain carrying a heteroallelic duplication of the ade6 gene was used to isolate mitotic recombination-deficient mutants. Recombination between the different copies of the ade6 gene can lead to Ade+ segregants. These are observed as growing papillae when colonies of a suitable size are replicated onto selective medium. We isolated mutants which show an altered papillation phenotype. With two exceptions, they exhibit a decrease in the frequency of mitotic recombination between the heteroalleles of the duplication. The two other mutants display a hyper-recombination phenotype. The 12 mutations were allocated to at least nine distinct loci by recombination tests. Of the eight rec mutants analyzed further, six were also affected in mitotic intergenic recombination in the intervals cen2-mat or cen3-arg 1. No effect on mitotic intragenic recombination was observed. These data suggest that mitotic gene conversion and crossing over can be separated mutationally. Meiotic recombination occurs at the wild-type frequency in all mutants investigated.     

In vivo human somatic mutation: frequency and spectrum with age.

The number and molecular nature of in vivo mutations in relation to age was studied at the autosomal HLA-A locus in human lymphocytes. Mutant lymphocytes were isolated by immunoselection, cloned at limiting dilution and enumerated, and the HLA-A gene and other polymorphic gene loci on chromosome 6 were studied by Southern blotting to determine gene dosage and loss of heterozygosity. Results of 167 assays in 73 individuals showed that the total number of mutant lymphocytes increased significantly with age from a geometric mean frequency of 0.71 x 10(-5) in neonates to 6.53 x 10(-5) in elderly individuals. Analysis of rearrangement of T lymphocyte receptor beta or gamma chain genes gave a best estimate of 3.3% for the proportion of mutant lymphocytes detected which are clonally related. Molecular study of 434 mutants from 31 individuals showed no change on Southern blotting in 64.7%, gene deletion in 2.8% and mitotic recombination in 32.5%. Two mutants due to gene conversion but no mutants due to non-disjunction were detected. The number of 'no change' and recombination mutants increased significantly with age. There was a significant difference between individuals in the proportion of mutants which resulted from mitotic recombination and the data suggested that the proportion was bimodally distributed. The point of crossing-over in recombination mutants was predominantly randomly distributed between the HLA-A locus and the centromere.     

The Effects of Insertions on Mammalian Intrachromosomal Recombination

We examined the effects of insertion mutations on intrachromosomal recombination. A series of mouse L cell lines carrying mutant herpes simplex virus thymidine kinase (tk) heteroalleles was generated; these lines differed in the nature of their insertion mutations. In direct repeat lines with different large insertions in each gene, there was a 20-fold drop in gene conversion rate and only a five-fold drop in crossover rate relative to the analogous rates in lines with small insertions in each gene. Surprisingly, in direct repeat lines carrying the same large insertion in each gene, there was a larger drop in both types of recombination. When intrachromosomal recombination between inverted repeat tk genes with different large insertions was examined, we found that the rate of gene conversion dropped five-fold relative to small insertions, while the rate of crossing over was unaffected. The differential effects on conversion and crossing over imply that gene conversion is more sensitive to insertion mutation size. Finally, the fraction of gene conversions associated with a crossover increased from 2% for inverted repeats with small insertions to 18% for inverted repeats with large insertions. One interpretation of this finding is that during intrachromosomal recombination in mouse cells long conversion tracts are more often associated with crossing over.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.