The role of CSM3, MRC1, and TOF1 in minisatellite stability and large loop DNA repair during meiosis in yeast.

Double-stranded break (DSB) repair during meiotic recombination in yeast Saccharomyces cerevisiae leads to the formation of heteroduplex DNA, a hybrid DNA molecule composed of single strands from two homologous chromosomes. Differences in sequence between the strands within heteroduplex DNA generate mismatches or large unpaired loops that are substrates for repair. At least two pathways function to repair large loops that form within heteroduplex DNA: the RAD1-dependent large loop repair (LLR) pathway and another as yet uncharacterized RAD1-independent LLR pathway. Repair of large loops during meiotic recombination is especially important for the genomic stability of the repetitive DNA sequences known as minisatellites. Minisatellite DNA tracts are generally stable during mitotic cell divisions but frequently alter in length during meiosis. Using a yeast minisatellite system in which the human minisatellite associated with the HRAS1 proto-oncogene has been inserted into the recombination hotspot region upstream of HIS4 in S. cerevisiae, our lab previously showed that the RAD1-dependent LLR pathway controls minisatellite length expansions, but not contractions. Here we show that minisatellite length expansions are controlled by the products of the CSM3 and TOF1 genes, while contractions are controlled by MRC1. By examining meiotic segregation patterns in yeast strains heterozygous for the 26bp his4-lopd insert, we found that deleting CSM3 caused a loss of LLR activity similar to that seen in a RAD1 mutant. Double mutant analysis revealed that failure to repair loops is exacerbated upon deleting both RAD1 and CSM3 - specifically the type of repair that fills in loops, which would generate minisatellite length expansions. A model for minisatellite length alteration based on these results is presented.

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans.

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom's syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition.

Haplotype mapping of a diploid non-meiotic organism using existing and induced aneuploidies.

Haplotype maps (HapMaps) reveal underlying sequence variation and facilitate the study of recombination and genetic diversity. In general, HapMaps are produced by analysis of Single-Nucleotide Polymorphism (SNP) segregation in large numbers of meiotic progeny. Candida albicans, the most common human fungal pathogen, is an obligate diploid that does not appear to undergo meiosis. Thus, standard methods for haplotype mapping cannot be used. We exploited naturally occurring aneuploid strains to determine the haplotypes of the eight chromosome pairs in the C. albicans laboratory strain SC5314 and in a clinical isolate. Comparison of the maps revealed that the clinical strain had undergone a significant amount of genome rearrangement, consisting primarily of crossover or gene conversion recombination events. SNP map haplotyping revealed that insertion and activation of the UAU1 cassette in essential and non-essential genes can result in whole chromosome aneuploidy. UAU1 is often used to construct homozygous deletions of targeted genes in C. albicans; the exact mechanism (trisomy followed by chromosome loss versus gene conversion) has not been determined. UAU1 insertion into the essential ORC1 gene resulted in a large proportion of trisomic strains, while gene conversion events predominated when UAU1 was inserted into the non-essential LRO1 gene. Therefore, induced aneuploidies can be used to generate HapMaps, which are essential for analyzing genome alterations and mitotic recombination events in this clonal organism.

Zinc regulates the stability of repetitive minisatellite DNA tracts during stationary phase.

Repetitive minisatellite DNA tracts are stable in mitotic cells but unstable in meiosis, altering in repeat number and repeat composition. As relatively little is known about the factors that influence minisatellite stability, we isolated mutations that destabilize a minisatellite repeat tract in the ADE2 gene of Saccharomyces cerevisiae. One mutant class exhibited a novel color segregation phenotype, "blebbing," characterized by minisatellite instability during stationary phase. Minisatellite tract alterations in blebbing strains consist exclusively of the loss of one 20-bp repeat. Timing experiments suggest that these tract alterations occur only after cells have entered stationary phase. Two complementation groups identified in this screen have mutations in either the high-affinity zinc transporter ZRT1 or its zinc-dependent transcriptional regulator ZAP1. The Deltazrt1 mutant specifically affects the stability of minisatellite tracts; microsatellites or simple insertions in the ADE2 reading frame are not destabilized by loss of ZRT1. The Deltazrt1 blebbing phenotype is partially dependent on a functional RAD50. Zinc is known for its role as an essential cofactor in many DNA-binding proteins. We describe possible models by which zinc can influence minisatellite stability. Our findings directly implicate zinc homeostasis in the maintenance of genomic stability during stationary phase.

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans.

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Delta/mre11Delta, rad50Delta/rad50Delta, and rad52Delta/rad52Delta mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Delta/msh2Delta, pms1Delta/pms1Delta, and yku80Delta/yku80Delta mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Delta/mre11Delta and rad50Delta/rad50Delta mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Delta/mre11Delta, rad50Delta/rad50Delta, or rad52Delta/rad52Delta leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Delta/msh2Delta and pms1Delta/pms1Delta (MMR mutants) and rad50Delta/rad50Delta (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.

RAD1 controls the meiotic expansion of the human HRAS1 minisatellite in Saccharomyces cerevisiae.

Minisatellite DNA is repetitive DNA with a repeat unit length from 15 to 100 bp. While stable during mitosis, it destabilizes during meiosis, altering both in length and in sequence composition. The basis for this instability is unknown. To investigate the factors controlling minisatellite stability, a minisatellite sequence 3' of the human HRAS1 gene was introduced into the Saccharomyces cerevisiae genome, replacing the wild-type HIS4 promoter. The minisatellite tract exhibited the same phenotypes in yeast that it exhibited in mammalian systems. The insertion stimulated transcription of the HIS4 gene; mRNA production was detected at levels above those seen with the wild-type promoter. The insertion stimulated meiotic recombination and created a hot spot for initiation of double-strand breaks during meiosis in the regions immediately flanking the repetitive DNA. The tract length altered at a high frequency during meiosis, and both expansions and contractions in length were detected. Tract expansion, but not contraction, was controlled by the product of the RAD1 gene. RAD1 is the first gene identified that controls specifically the expansion of minisatellite tracts. A model for tract length alteration based on these results is presented.

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

Length and sequence heterozygosity differentially affect HRAS1 minisatellite stability during meiosis in yeast.

Minisatellites, one of the major classes of repetitive DNA sequences in eukaryotic genomes, are stable in somatic cells but destabilize during meiosis. We previously established a yeast model system by inserting the human Ha-ras/HRAS1 minisatellite into the HIS4 promoter and demonstrated that our system recapitulates all of the phenotypes associated with the human minisatellite. Here we demonstrate that meiotic minisatellite tract-length changes are half as frequent in diploid cells harboring heterozygous HRAS1 minisatellite tracts in which the two tracts differ by only two bases when compared to a strain with homozygous minisatellite tracts. Further, this decrease in alteration frequency is entirely dependent on DNA mismatch repair. In contrast, in a diploid strain containing heterozygous minisatellite tract alleles differing in length by three complete repeats, length alterations are observed at twice the frequency seen in a strain with homozygous tracts. Alterations consist of previously undetectable gene conversion events, plus nonparental length alteration events seen previously in strains with homozygous tracts. A strain containing tracts with both base and length heterozygosity exhibits the same level of alteration as a strain containing only length heterozygosity, indicating that base heterozygosity-dependent tract stabilization does not affect tract-length alterations occurring by gene conversion.

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis.

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.

Novel checkpoint pathway organization promotes genome stability in stationary-phase yeast cells.

Most DNA alterations occur during DNA replication in the S phase of the cell cycle. However, the majority of eukaryotic cells exist in a nondividing, quiescent state. Little is known about the factors involved in preventing DNA instability within this stationary-phase cell population. Previously, we utilized a unique assay system to identify mutations that increased minisatellite alterations specifically in quiescent cells in Saccharomyces cerevisiae. Here we conducted a modified version of synthetic genetic array analysis to determine if checkpoint signaling components play a role in stabilizing minisatellites in stationary-phase yeast cells. Our results revealed that a subset of checkpoint components, specifically MRC1, CSM3, TOF1, DDC1, RAD17, MEC3, TEL1, MEC1, and RAD53, prevent stationary-phase minisatellite alterations within the quiescent cell subpopulation of stationary-phase cells. Pathway analysis revealed at least three pathways, with MRC1, CSM3, and TOF1 acting in a pathway independent of MEC1 and RAD53. Overall, our data indicate that some well-characterized checkpoint components maintain minisatellite stability in stationary-phase cells but are regulated differently in those cells than in actively growing cells. For the MRC1-dependent pathway, the checkpoint itself may not be the important element; rather, it may be loss of the checkpoint proteins' other functions that contributes to DNA instability.

A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells.

Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15-100 bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary-phase cells. Alterations within minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy, and several types of cancer. However, little is known about the factors preventing minisatellite alterations. Previously, our laboratory developed a color segregation assay in which a minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that minisatellite alterations that occur in stationary-phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the synthetic genetic array analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different minisatellite tracts: the ade2-min3 construct consisting of three identical 20-bp repeats, and the ade2-h7.5 construct, consisting of seven-and-a-half 28-bp variable repeats. Mutations in 102 and 157 genes affect the stability of the ade2-min3 and ade2-h7.5 alleles, respectively. Only seven hits overlapped both screens, indicating that different factors regulate repeat stability depending upon minisatellite size and composition. Importantly, we demonstrate that mismatch repair influences the stability of the ade2-h7.5 allele, indicating that this type of DNA repair stabilizes complex minisatellites in stationary phase cells. Our work provides insight into the factors regulating minisatellite stability.

Multiple pathways regulate minisatellite stability during stationary phase in yeast.

Alterations in minisatellite DNA repeat tracts in humans have been correlated with a number of serious disorders, including cancer. Despite their importance for human health, the genetic factors that influence minisatellite stability are not well understood. Previously, we identified mutations in the Saccharomyces cerevisiae zinc homeostasis genes ZRT1 and ZAP1 that significantly increase the frequency of minisatellite alteration specifically during stationary phase. In this work, we identified mutants of END3, PKC1, and RAD27 that increase minisatellite instability during stationary phase. Genetic analysis reveals that these genes, along with ZRT1 and ZAP1, comprise multiple pathways regulating minisatellite stability during stationary phase. Minisatellite alterations generated by perturbation of any of these pathways occur via homologous recombination. We present evidence that suggests formation of ssDNA or ssDNA breaks may play a primary role in stationary phase instability. Finally, we examined the roles of these pathways in the stability of a human minisatellite tract associated with the HRAS1 oncogene and found that loss of RAD27, but not END3 or PKC1, destabilizes the HRAS1 minisatellite in stationary phase yeast. This result indicates that the genetic control of stationary phase minisatellite stability is dependent on the sequence composition of the minisatellite itself.

Minisatellite alterations in ZRT1 mutants occur via RAD52-dependent and RAD52-independent mechanisms in quiescent stationary phase yeast cells.

Alterations in minisatellite DNA repeat tracts are associated with a variety of human diseases including Type 1 diabetes, progressive myoclonus epilepsy, and some types of cancer. However, in spite of their role in human health, the factors required for minisatellite alterations are not well understood. We previously identified a stationary phase specific increase in minisatellite instability caused by mutations in the high affinity zinc transporter ZRT1, using a minisatellite inserted into the ADE2 locus in Saccharomyces cerevisiae. Here, we examined ZRT1-mediated minisatellite instability in yeast strains lacking key recombination genes to determine the mechanisms by which these alterations occur. Our analysis revealed that minisatellite alterations in a Δzrt1 mutant occur by a combination of RAD52-dependent and RAD52-independent mechanisms. In this study, plasmid-based experiments demonstrate that ZRT1-mediated minisatellite alterations occur independently of chromosomal context or adenine auxotrophy, and confirmed the stationary phase timing of the events. To further examine the stationary phase specificity of ZRT1-mediated minisatellite alterations, we deleted ETR1 and POR1, genes that were previously shown to differentially affect the viability of quiescent or nonquiescent cells in stationary phase populations. These experiments revealed that minisatellite alterations in Δzrt1 mutants occur exclusively in quiescent stationary phase cells. Finally, we show that loss of ZRT1 stimulates alterations in a derivative of the human HRAS1 minisatellite. We propose that the mechanism of ZRT1-mediated minisatellite instability during quiescence is relevant to human cells, and thus, human disease.

Analysis of base excision and nucleotide excision repair in Candida albicans.

Candida albicans, clinically the most important human fungal pathogen, rapidly develops resistance to antifungal drugs. The acquisition of resistance has been linked to various types of genome changes. As part of an ongoing study of this problem, we investigated mutation, genome stability and drug resistance acquisition in C. albicans strains with deletions in the base excision repair (BER) genes NTG1, APN1 and OGG1, and in the nucleotide excision repair (NER) genes RAD2 and RAD10. The BER mutants did not exhibit any change in their susceptibility to DNA-damaging agents, but the NER mutants were extremely sensitive to UV-induced DNA damage. We did not observe any significant change in mutation, genome stability and antifungal drug sensitivity in the mutant strains we tested. However, we detected a number of intriguing phenotypic differences between strains bearing deletions in equivalent C. albicans and Saccharomyces cerevisiae BER and NER genes, which may be related to differences in the life cycles of these two fungi.

A novel yeast genomic DNA library on a geneticin-resistance vector.

We have constructed a Saccharomyces cerevisiae genomic library utilizing a novel centromere-containing vector bearing the TRP1 and KANMX4 G418-resistance selectable markers. Random Sau3AI genomic DNA fragments with an average size of 6.4 kb were introduced into a unique BamHI site in the vector's polylinker. Six independent library pools were generated and the characteristics of these pools were determined. This library can be used to introduce low-copy genomic DNA fragments into any strain that is not G418-resistant, making it useful for low-copy overexpression analysis or complementation of mutant phenotypes in strains that lack a suitable auxotrophy marker or only exhibit phenotypes on rich, undefined, growth media.