Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Delta double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Delta alone. However, surprisingly, the dna2-2 sgs1Delta lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Delta lethality is only partially suppressed by deletion of rad51Delta. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.     

Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with deltarrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.     

The pattern of sensitivity of yeast dna2 mutants to DNA damaging agents suggests a role in DSB and postreplication repair pathways

The Saccharomyces cerevisiae DNA2 gene encodes a DNA-stimulated ATPase and DNA helicase/nuclease essential for DNA replication. In characterizing dna2 mutants, we have found that Dna2p also participates in DNA repair or in damage avoidance mechanisms. dna2 mutants are sensitive to X rays, although they are less sensitive than rad52 mutants. The X-ray sensitivity of dna2 mutants is suppressed by overexpression of a 5' to 3' exonuclease, the yeast FEN-1 structure-specific nuclease, encoded by the RAD27 gene, which also suppresses the growth defect of dna2-ts mutants. SGS1 encodes a helicase with similar properties to Dna2 protein. Although sgs1Delta mutants are resistant to X rays, dna2-2 sgs1Delta double mutants are more sensitive to X rays than the dna2-2 mutant. Temperature sensitive dna2 mutants are only slightly sensitive to UV light, show normal levels of spontaneous and UV induced mutagenesis, and have only a 2.5-fold elevated level of dinucleotide tract instability compared to wildtype. However, dna2Delta strains kept alive by overproduction of RAD27 are highly sensitive to UV light. These phenotypes, in addition to the epistasis analysis reported, allow us to propose that Dna2 is involved in postreplication and DSB repair pathways.     

Human premature aging, DNA repair and RecQ helicases

Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.     

Biochemical characterization of the WRN-1 RecQ helicase of Caenorhabditis elegans

The highly conserved RecQ helicases are essential for the maintenance of genomic stability. Werner syndrome protein, WRN, is one of five human RecQ helicase homologues, and a deficiency of the protein causes a hereditary premature aging disorder that is characterized by genomic instability. A WRN orthologue, wrn-1 lacking the exonuclease domain, has been identified in the nematode Caenorhabditis elegans. wrn-1(RNAi) in C. elegans has a shortened life span, increased sensitivity to DNA damage, and accelerated aging phenotypes. However, little is known about its enzymatic activity. We purified the recombinant C. elegans WRN-1 protein (CeWRN-1) and then investigated its substrate specificity in vitro to improve our understanding of its function in vivo. We found that CeWRN-1 is an ATP-dependent 3'-5' helicase capable of unwinding a variety of DNA structures such as forked duplexes, Holliday junctions, bubble substrates, D-loops, and flap duplexes, and 3'-tailed duplex substrates. Distinctly, CeWRN-1 is able to unwind a long forked duplex compared to human WRN. Furthermore, CeWRN-1 helicase activity on a long DNA duplex is stimulated by C. elegans replication protein A (CeRPA) that is shown to interact with CeWRN-1 by a dot blot. The ability of CeWRN-1 to unwind these DNA structures may improve the access for DNA repair and replication proteins that are important for preventing the accumulation of abnormal structures, contributing to genomic stability.     

A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.

We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication. We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase. FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in Okazaki fragment maturation. Overproduction of yFEN-1, encoded by RAD27/RTH1, suppresses the temperature-sensitive growth of dna2-1 mutants. Overproduction of Dna2 suppresses the rad27/rth1 delta temperature-sensitive growth defect. dna2-1 rad27/rth1 delta double mutants are inviable, indicating that the mutations are synthetically lethal. The genetic interactions are likely due to direct physical interaction between the two proteins, since both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2. The simplest interpretation of these data is that one of the roles of Dna2 helicase is associated with processing of Okazaki fragments.     

The short life span of Saccharomyces cerevisiae sgs1 and srs2 mutants is a composite of normal aging processes and mitotic arrest due to defective recombination

Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.     

Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases

Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere maintenance including genomic instability, premature senescence, and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends.     

RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli

After UV irradiation, recA mutants fail to recover replication, and a dramatic and nearly complete degradation of the genomic DNA occurs. Although the RecBCD helicase/nuclease complex is known to mediate this catastrophic DNA degradation, it is not known how or where this degradation is initiated. Previous studies have speculated that RecBCD targets and initiates degradation from the nascent DNA at replication forks arrested by DNA damage. To test this question, we examined which enzymes were responsible for the degradation of genomic DNA and the nascent DNA in UV-irradiated recA cells. We show here that, although RecBCD degrades the genomic DNA after UV irradiation, it does not target the nascent DNA at arrested replication forks. Instead, we observed that the nascent DNA at arrested replication forks in recA cultures is degraded by RecJ/RecQ, similar to what occurs in wild-type cultures. These findings indicate that the genomic DNA degradation and nascent DNA degradation in UV-irradiated recA mutants are mediated separately through RecBCD and RecJ/RecQ, respectively. In addition, they demonstrate that RecBCD initiates degradation at a site(s) other than the arrested replication fork directly.     

Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence

Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures.     

hpr1Δ Affects Ribosomal DNA Recombination and Cell Life Span in Saccharomyces cerevisiae

Multiple genetic pathways have been shown to regulate life span and aging in the yeast Saccharomyces cerevisiae. Here we show that loss of a component of the RNA polymerase II complex, Hpr1p, results in a decreased life span. Although hpr1Delta mutants have an increased rate of recombination within the ribosomal DNA (rDNA) array, this is not accompanied by an increase in extrachromosomal rDNA circles (ERCs). Analyses of mutants that affect replication of the rDNA array and suppressors that reverse the phenotypes of the hpr1Delta mutant show that the reduced life span is associated with increased genomic instability but not with increased ERC formation. The hpr1Delta mutant acts in a pathway distinct from previously described mutants that reduce life span.     

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.     

Longevity mutation in SCH9 prevents recombination errors and premature genomic instability in a Werner/Bloom model system

Werner and Bloom syndromes are human diseases characterized by premature age-related defects including elevated cancer incidence. Using a novel Saccharomyces cerevisiae model system for aging and cancer, we show that cells lacking the RecQ helicase SGS1 (WRN and BLM homologue) undergo premature age-related changes, including reduced life span under stress and calorie restriction (CR), G1 arrest defects, dedifferentiation, elevated recombination errors, and age-dependent increase in DNA mutations. Lack of SGS1 results in a 110-fold increase in gross chromosomal rearrangement frequency during aging of nondividing cells compared with that generated during the initial population expansion. This underscores the central role of aging in genomic instability. The deletion of SCH9 (homologous to AKT and S6K), but not CR, protects against the age-dependent defects in sgs1Δ by inhibiting error-prone recombination and preventing DNA damage and dedifferentiation. The conserved function of Akt/S6k homologues in lifespan regulation raises the possibility that modulation of the IGF-I–Akt–56K pathway can protect against premature aging syndromes in mammals.     

The Rb/E2F pathway and Ras activation regulate RecQ helicase gene expression

Disruption of the Rb (retinoblastoma protein)/E2F cell-cycle pathway and Ras activation are two of the most frequent events in cancer, and both of these mutations place oncogenic stress on cells to increase DNA replication. In the present study, we demonstrate that these mutations have an additive effect on induction of members of the RecQ DNA helicase family. RecQ activity is important for genomic stability, initiation of DNA replication and telomere maintenance, and mutation of the BLM (Bloom's syndrome gene), WRN (Werner's syndrome gene) or RECQL4 (Rothmund-Thomson syndrome gene) family members leads to premature aging syndromes characterized by genetic instability and telomere loss. RecQ family members are frequently overexpressed in cancers, and overexpression of BLM has been shown to cause telomere elongation. Concomitant with induction of RecQ genes in response to Rb family mutation and Ras activation, we show an increase in the number of telomeric repeats. We suggest that this induction of RecQ genes in response to common oncogenic mutations may explain the up-regulation of the genes seen in cancers, and it may provide a means for transformed cells to respond to an increased demand for DNA replication.     

Compromised cellular responses to DNA damage accelerate chronological aging by incurring cell wall fragility in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Elevated levels of reactive oxygen species (ROS) can attack almost all cell components including genomic DNA to induce many types of DNA damage. In this study, we used Saccharomyces cerevisiae with various mutations in a biological network supposed to prevent deleterious effects of endogenous ROS to test the effect of such a network on yeast chronological aging. Our results showed that cells with defects in cellular antioxidation, DNA repair and DNA damage checkpoints displayed a mutation rate higher than that of wild-type strain. Moreover, the chronological life span of most mutants as determined by colony formation was found to be shorter than that of wild-type cells, especially for the mutants defective in DNA replication and DNA damage checkpoints, although the observed cell number was almost the same for wild-type and mutant strains. The mutants were finally found to be more sensitive to SDS and lysing enzyme treatment, and that the degree of sensitivity was correlated with their chronological life span.     

Characterization of the slow-growth phenotype of S. cerevisiae Whip/Mgs1 Sgs1 double deletion mutants

RecQ DNA helicases from many organisms have been indicated to function in the maintenance of genomic stability. In human cells, mutation in the WRN helicase, a RecQ-like DNA helicase, results in the Werner syndrome (WS), a genetic disorder characterized by genomic instability and premature ageing. Similarly, mutation in SGS1, the RECQ homologue in budding yeast, results in genomic instability and accelerated ageing. We previously demonstrated that mouse WRN interacts physically with a novel, highly conserved protein that we named WHIP, and that in budding yeast cells, simultaneous deletion of WHIP/MGS1 and SGS1 results in slow growth and shortened life span. Here we show by using genetic analysis in Saccharomyces cerevisiae that mgs1Delta sgs1Delta cells have increased rates of terminal G2/M arrest, and show elevated rates of spontaneous sister chromatid recombination (SCR) and rDNA array recombination. Finally, we report that complementation of the synthetic relationship between SGS1 and WHIP/MGS1 requires both the helicase and Top3-binding activities of Sgs1, as well as the ATPase activity of Mgs1. Our results suggest that Whip/Mgs1 is implicated in DNA metabolism, and is required for normal growth and cell cycle progression in the absence of Sgs1.     

Genomic instability is associated with natural life span variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for approximately 50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span.     

The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2

Werner syndrome (WS) is characterized by features of premature aging and is caused by loss of the RecQ helicase protein WRN. WS fibroblasts display defects associated with telomere dysfunction, including accelerated telomere erosion and premature senescence. In yeast, RecQ helicases act in an alternative pathway for telomere lengthening (ALT) via homologous recombination. We found that WRN associates with telomeres when dissociation of telomeric D loops is likely during replication and recombination. In human ALT cells, WRN associates directly with telomeric DNA. The majority of TRF1/PCNA colocalizing foci contained WRN in live S phase ALT cells but not in telomerase-positive HeLa cells. Biochemically, the WRN helicase and 3' to 5' exonuclease act simultaneously and cooperate to release the 3' invading tail from a telomeric D loop in vitro. The telomere binding proteins TRF1 and TRF2 limit digestion by WRN. We propose roles for WRN in dissociating telomeric structures in telomerase-deficient cells.     

Delineation of WRN helicase function with EXO1 in the replicational stress response

The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50-Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS). WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom's syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases.