The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.     

A transient α-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1

The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays show that the defects of the proline mutants are the result of impaired binding of Top3 and Rmi1 to Sgs1. Extending mutagenesis N-terminally revealed a second functionally critical region that spans residues 9–17. Depending on the position of the proline substitution in the helix functional impairment of Sgs1 function varied, gradually increasing from the C- to the N-terminus. The multiscale approach we used to interrogate structure/function relationships in the long disordered N-terminal segment of Sgs1 allowed us to precisely define a functionally critical region and should be generally applicable to other disordered proteins.     

The possible roles of the DNA helicase and C-terminal domains in RECQ5/QE: complementation study in yeast

DmRECQ5/QE is a member of the RECQ5 subfamily, which shares homology with the Escherichia coli RecQ DNA helicase. Although the DNA helicase activity of RECQ5/QE has been characterized in vitro, the in vivo function of RECQ5/QE was essentially unknown. To investigate the cellular role of RECQ5, the potential of RECQ5/QE was evaluated by substitution of the only RecQ-like helicase, Sgs1, in budding yeast. RECQ5/QE can complement several phenotypes of sgs1, including the synthetic growth defect with srs2, the hypersensitivity to hydroxyurea and methyl methanesulfonate, and the elevated frequency of homologous recombination and sister chromatid exchange (SCE), but poorly complemented the suppression of slow growth in top3. These data suggested that RECQ5/QE exhibits an evolutionarily conserved RecQ function in vivo. The RECQ5/QE domain necessary for the yeast complementation was determined. The helicase domain and helicase activity were required to complement both the sgs1srs2 and sgs1top3 phenotypes. In contrast, the C-terminal domain was dispensable for complementing the sgs1srs2 phenotype, but was required for the sgs1top3 phenotype. These results suggested that the RECQ5/QE helicase activity is important for cellular function and that the C-terminal domain has a specific function in the absence of Top3.     

Disruption of SUMO-targeted ubiquitin ligases Slx5–Slx8/RNF4 alters RecQ-like helicase Sgs1/BLM localization in yeast and human cells

RecQ-like helicases are a highly conserved protein family that functions during DNA repair and, when mutated in humans, is associated with cancer and/or premature aging syndromes. The budding yeast RecQ-like helicase Sgs1 has important functions in double-strand break (DSB) repair of exogenously induced breaks, as well as those that arise endogenously, for example during DNA replication. To further investigate Sgs1's regulation, we analyzed the subcellular localization of a fluorescent fusion of Sgs1 upon DNA damage. Consistent with a role in DSB repair, Sgs1 recruitment into nuclear foci in asynchronous cultures increases after ionizing radiation (IR) and after exposure to the alkylating agent methyl methanesulfonate (MMS). Yet, despite the importance of Sgs1 in replicative damage repair and in contrast to its elevated protein levels during S-phase, we find that the number of Sgs1 foci decreases upon nucleotide pool depletion by hydroxyurea (HU) treatment and that this negative regulation depends on the intra S-phase checkpoint kinase Mec1. Importantly, we identify the SUMO-targeted ubiquitin ligase (STUbL) complex Slx5–Slx8 as a negative regulator of Sgs1 foci, both spontaneously and upon replicative damage. Slx5–Slx8 regulation of Sgs1 foci is likely conserved in eukaryotes, since expression of the mammalian Slx5–Slx8 functional homologue, RNF4, restores Sgs1 focus number in slx8 cells and furthermore, knockdown of RNF4 leads to more BLM foci in U-2 OS cells. Our results point to a model where RecQ-like helicase subcellular localization is regulated by STUbLs in response to DNA damage, presumably to prevent illegitimate recombination events.     

The role of post-translational modifications in fine-tuning BLM helicase function during DNA repair

RecQ-like helicases are a highly conserved family of proteins which are critical for preserving genome integrity. Genome instability is considered a hallmark of cancer and mutations within three of the five human RECQ genes cause hereditary syndromes that are associated with cancer predisposition. The human RecQ-like helicase BLM has a central role in DNA damage signaling, repair, replication, and telomere maintenance. BLM and its budding yeast orthologue Sgs1 unwind double-stranded DNA intermediates. Intriguingly, BLM functions in both a pro- and anti-recombinogenic manner upon replicative damage, acting on similar substrates. Thus, BLM activity must be intricately controlled to prevent illegitimate recombination events that could have detrimental effects on genome integrity. In recent years it has become evident that post-translational modifications (PTMs) of BLM allow a fine-tuning of its function. To date, BLM phosphorylation, ubiquitination, and SUMOylation have been identified, in turn regulating its subcellular localization, protein–protein interactions, and protein stability. In this review, we will discuss the cellular context of when and how these different modifications of BLM occur. We will reflect on the current model of how PTMs control BLM function during DNA damage repair and compare this to what is known about post-translational regulation of the budding yeast orthologue Sgs1. Finally, we will provide an outlook toward future research, in particular to dissect the cross-talk between the individual PTMs on BLM.     

Different domains of Sgs1 are required for mitotic and meiotic functions

The SGS1 of Saccharomyces cerevisiae is a homologue for human Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome causative genes. Disruptants of SGS1 show high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea, and hyper recombination phenotypes including interchromosomal homologous recombination in mitotic growth. In addition, sgs1 disruptants show poor sporulation and a reduced level of meiotic recombination as assayed by return-to-growth. We examined domains of Sgs1 required for mitotic and meiotic functions of Sgs1 by transfecting variously mutated SGS1 into sgs1 disruptants. The N-terminal 1-401 amino acid region was required for complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations of sgs1 disruptants in mitotic growth and for complementation of poor sporulation and of reduced meiotic recombination. Although the N-terminal 1-125 amino acid region was absolutely required for the complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations in mitotic growth, it was dispensable for the meiotic functions. In contrast, the highly acidic region (400-596 amino acid) was dispensable for the mitotic functions but a deletion of this region affected the meiotic functions. The C-terminal 1271-1350 amino acid region containing a HRDC (helicase and RNaseD C-terminal) domain was dispensable for the mitotic and meiotic functions. Although DNA helicase activity of Sgs1 was not required for Sgs1 to complement the meiotic functions, a deletion of helicase motifs III-IV (842-1046 amino acid) abolished the complementing activity of Sgs1, indicating that a structurally intact helicase domain is necessary for Sgs1 to fulfill its meiotic functions.     

SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases

Sgs1 is a member of the RecQ family of DNA helicases, which have been implicated in genomic stability, cancer and ageing. Srs2 is another DNA helicase that shares several phenotypic features with Sgs1 and double sgs1srs2 mutants have a severe synthetic growth phenotype. This suggests that there may be functional overlap between these two DNA helicases. Consistent with this idea, we found the srs2Δ mutant to have a similar genotoxin sensitivity profile and replicative lifespan to the sgs1Δ mutant. In order to directly test if Sgs1 and Srs2 are functionally interchangeable, the ability of high-copy SGS1 and SRS2 plasmids to complement the srs2Δ and sgs1Δ mutants was assessed. We report here that SGS1 is a multicopy suppressor of the methyl methanesulphonate (MMS) and hydroxyurea sensitivity of the srs2Δ mutant, whereas SRS2 overexpression had no complementing ability in the sgs1Δ mutant. Domains of Sgs1 directly required for processing MMS-induced DNA damage, most notably the helicase domain, are also required for complementation of the srs2Δ mutant. Although SGS1 overexpression was unable to rescue the shortened mean replicative lifespan of the srs2Δ mutant, maximum lifespan was significantly increased by multicopy SGS1. We conclude that Sgs1 is able to partially compensate for the loss of Srs2.     

Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases

BLM and WRN are members of the RecQ family of DNA helicases that act to suppress genome instability and cancer predisposition. In addition to a RecQ helicase domain, each of these proteins contains an N-terminal domain of approximately 500 amino acids (aa) that is incompletely characterized. Previously, we showed that the N-terminus of Sgs1, the yeast ortholog of BLM, contains a physiologically important 200 aa domain (Sgs1_103–322) that displays single-stranded DNA (ssDNA) binding, strand annealing (SA), and apparent strand-exchange (SE) activities in vitro. Here we used a genetic assay to search for heterologous proteins that could functionally replace this domain of Sgs1 in vivo. In contrast to Rad59, the oligomeric Rad52 protein provided in vivo complementation, suggesting that multimerization is functionally important. An N-terminal domain of WRN was also identified that could replace Sgs1_103–322 in yeast. This domain, WRN_235–526, contains a known coiled coil and displays the same SA and SE activities as Sgs1_103–322. The coiled coil domain of WRN_235–526 is required for both its in vivo activity and its in vitro SE activity_. Based on this result, a potential coiled coil was identified within Sgs1_103–322. This 25 amino acid region was similarly essential for wt Sgs1 activity in vivo and was replaceable by a heterologous coiled coil. Taken together, the results indicate that a coiled coil and a closely linked apparent SE activity are conserved features of the BLM and WRN DNA helicases.     

An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks

DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase α at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8.     

Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1

The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.     

Sumoylation of the BLM ortholog,Sgs1, promotes telomere–telomere recombination in budding yeast

BLM and WRN are members of the RecQ family of DNA helicases, and in humans their loss is associated with syndromes characterized by genome instability and cancer predisposition. As the only RecQ DNA helicase in the yeast Saccharomyces cerevisiae, Sgs1 is known to safeguard genome integrity through its role in DNA recombination. Interestingly, WRN, BLM and Sgs1 are all known to be modified by the small ubiquitin-related modifier (SUMO), although the significance of this posttranslational modification remains elusive. Here, we demonstrate that Sgs1 is specifically sumoylated under the stress of DNA double strand breaks. The major SUMO attachment site in Sgs1 is lysine 621, which lies between the Top3 binding domain and the DNA helicase domain. Surprisingly, sumoylation of K621 was found to be uniquely required for Sgs1’s role in telomere–telomere recombination. In contrast, sumoylation was dispensable for Sgs1’s roles in DNA damage tolerance, supppression of direct repeat and rDNA recombination, and promotion of top3Δ slow growth. Our results demonstrate that although modification by SUMO is a conserved feature of RecQ family DNA helicases, the major sites of modification are located on different domains of the protein in different organisms. We suggest that sumoylation of different domains of RecQ DNA helicases from different organisms contributes to conserved roles in regulating telomeric recombination.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence

RecQ DNA helicases, including yeast Sgs1p and the human Werner and Bloom syndrome proteins, participate in telomere biology, but the underlying mechanisms are not fully understood. Here, we explore the protein sequences and genetic interactors of Sgs1p that function to slow the senescence of telomerase (tlc1) mutants. We find that the S-phase checkpoint function of Sgs1p is dispensable for preventing rapid senescence, but that Sgs1p sequences required for homologous recombination, including the helicase domain and topoisomerase III interaction domain, are essential. sgs1 and rad52 mutations are epistatic during senescence, indicating that Sgs1p participates in a RAD52-dependent recombinational pathway of telomere maintenance. Several mutations that are synthetically lethal with sgs1 mutation and which individually lead to genome instability, including mus81, srs2, rrm3, slx1 and top1, do not speed the senescence of tlc1 mutants, indicating that the rapid senescence of sgs1 tlc1 mutants is not caused by generic genome instability. However, mutations in SLX5 or SLX8, which encode proteins that function together in a complex that is required for viability in sgs1 mutants, do speed the senescence of tlc1 mutants. These observations further define roles for RecQ helicases and related proteins in telomere maintenance.     

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over

BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability. RESULTS: We have identified novel functions for Sgs1 in meiosis. Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation. In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis. Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion. The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase. A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation. CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions. The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis. Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange. We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.     

The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1

Sgs1, the RecQ helicase homolog, and Top3, the type-IA topoisomerase, physically interact and are required for genomic stability in budding yeast. Similarly, topoisomerase III genes physically pair with homologs of SGS1 in humans that are involved in the cancer predisposition and premature aging diseases Bloom, Werner, and Rothmund-Thompson syndromes. In the absence of Top1 activity, sgs1 mutants are severely growth impaired. Here, we investigate the role of Sgs1 helicase activity and its N-terminal Top3 interaction domain by using an allele-replacement technique to integrate mutant alleles at the native SGS1 genomic locus. We compare the phenotype of helicase-defective (sgs1-hd) and N-terminal deletion (sgs1-NDelta) strains to wild-type and sgs1 null strains. Like the sgs1 null, sgs1-hd mutations suppress top3 slow growth, cause a growth defect in the absence of Srs2 helicase, and impair meiosis. However, for recombination and the synthetic interaction with top1Delta mutations, loss of helicase activity exhibits a less severe phenotype than the null. Interestingly, deletion of the Top3 interaction domain of Sgs1 causes a top3-like phenotype, and furthermore, this effect is dependent on helicase activity. These results suggest that the protein-protein interaction between these two DNA-metabolism enzymes, even in the absence of helicase activity, is important for their function in catalyzing specific changes in DNA topology.     

Bipartite structure of the SGS1 DNA helicase in Saccharomyces cerevisiae

SGS1 in yeast encodes a DNA helicase with homology to the human BLM and WRN proteins. This group of proteins is characterized by a highly conserved DNA helicase domain homologous to Escherichia coli RecQ and a large N-terminal domain of unknown function. To determine the role of these domains in SGS1 function, we constructed a series of truncation and helicase-defective (-hd) alleles and examined their ability to complement several sgs1 phenotypes. Certain SGS1 alleles showed distinct phenotypes: sgs1-hd failed to complement the MMS hypersensitivity and hyper-recombination phenotypes, but partially complemented the slow-growth suppression of top3 sgs1 strains and the top1 sgs1 growth defect. Unexpectedly, an allele that encodes the amino terminus alone showed essentially complete complementation of the hyper-recombination and top1 sgs1 defects. In contrast, an allele encoding the helicase domain alone was unable to complement any sgs1 phenotype. Small truncations of the N terminus resulted in hyper-recombination and slow-growth phenotypes in excess of the null allele. These hypermorphic phenotypes could be relieved by deleting more of the N terminus, or in some cases, by a point mutation in the helicase domain. Intragenic complementation experiments demonstrate that both the amino terminus and the DNA helicase are required for full SGS1 function. We conclude that the amino terminus of Sgs1 has an essential role in SGS1 function, distinct from that of the DNA helicase, with which it genetically interacts.     

Hrq1 functions independently of Sgs1 to preserve genome integrity in Saccharomyces cerevisiae

Maintenance of genome stability in eukaryotes involves a number of conserved proteins, including RecQ helicases, which play multiple roles at various steps in homologous recombination and DNA repair pathways. Sgs1 has been described as the only RecQ helicase in lower eukaryotes. However, recent studies revealed the presence of a second RecQ helicase, Hrq1, which is most homologous to human RECQL4. Here we show that hrq1Δ mutation resulted in increased mitotic recombination and spontaneous mutation in Saccharomyces cerevisiae, and sgs1Δ mutation had additive effects on the phenotypes of hrq1Δ. We also observed that the hrq1Δ mutant was sensitive to 4-nitroquinoline 1-oxide and cisplatin, which was not complemented by overexpression of Sgs1. In addition, the hrq1Δ sgs1Δ double mutant displayed synthetic growth defect as well as a shortened chronological life span compared with the respective single mutants. Analysis of the type of age-dependent Can^r mutations revealed that only point mutations were found in hrq1Δ, whereas significant numbers of gross deletion mutations were found in sgs1Δ. Our results suggest that Hrq1 is involved in recombination and DNA repair pathways in S. cerevisiae independent of Sgs1.     

G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition

To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM–G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.     

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.