The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication

The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild‐type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Δ cells. Unexpectedly, we find that these cells progress faster through S phase than their wild‐type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Δ cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase.

[1]  R. Rothstein,et al.  Holliday Junctions Accumulate in Replication Mutants via a RecA Homolog-Independent Mechanism , 1997, Cell.

[2]  H. Klein,et al.  hpr1Δ Affects Ribosomal DNA Recombination and Cell Life Span in Saccharomyces cerevisiae , 2002, Molecular and Cellular Biology.

[3]  T. Kobayashi,et al.  A yeast gene product, Fob1 protein, required for both replication fork blocking and recombinational hotspot activities , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[4]  S Povey,et al.  Dynamic molecular combing: stretching the whole human genome for high-resolution studies. , 1997, Science.

[5]  C. Jaulin,et al.  Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase , 2000, Oncogene.

[6]  Jin-Qiu Zhou,et al.  The Saccharomyces Pif1p DNA Helicase and the Highly Related Rrm3p Have Opposite Effects on Replication Fork Progression in Ribosomal DNA , 2000, Cell.

[7]  K. Tatebayashi,et al.  Bloom's syndrome gene suppresses premature ageing caused by Sgs1 deficiency in yeast , 1999, Genes to cells : devoted to molecular & cellular mechanisms.

[8]  Kyungjae Myung,et al.  Maintenance of Genome Stability in Saccharomyces cerevisiae , 2002, Science.

[9]  J. Sogo,et al.  Architecture of the Replication Fork Stalled at the 3′ End of Yeast Ribosomal Genes , 2000, Molecular and Cellular Biology.

[10]  M. Poot,et al.  Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid cell lines. , 1992, Experimental cell research.

[11]  L. Loeb,et al.  Human Werner Syndrome DNA Helicase Unwinds Tetrahelical Structures of the Fragile X Syndrome Repeat Sequence d(CGG) n * , 1999, The Journal of Biological Chemistry.

[12]  A Bensimon,et al.  Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains. , 2001, Nucleic acids research.

[13]  O. Hyrien.  Mechanisms and consequences of replication fork arrest. , 2000, Biochimie.

[14]  D. Botstein,et al.  A group of interacting yeast DNA replication genes. , 1991, Genes & Development.

[15]  S. Elledge,et al.  BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. , 2000, Genes & development.

[16]  M. McVey,et al.  The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase , 2001, The EMBO journal.

[17]  Etienne Schwob,et al.  The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1). , 2002, Molecular cell.

[18]  E. Louis,et al.  Sgs1: A eukaryotic homolog of E. coil RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation , 1995, Cell.

[19]  P. Leder,et al.  The Werner Syndrome Gene Product Co-purifies with the DNA Replication Complex and Interacts with PCNA and Topoisomerase I* , 1999, The Journal of Biological Chemistry.

[20]  J. German,et al.  A retarded rate of DNA chain growth in Bloom's syndrome. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Gasser,et al.  The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci. , 2000, Genes & development.

[22]  M. Lopes,et al.  Fork Reversal and ssDNA Accumulation at Stalled Replication Forks Owing to Checkpoint Defects , 2002, Science.

[23]  J. Diffley,et al.  Activation of dormant origins of DNA replication in budding yeast. , 1999, Genes & development.

[24]  J. Wang,et al.  Interaction between yeast sgs1 helicase and DNA topoisomerase III. , 2000, The Journal of biological chemistry.

[25]  I. Hickson,et al.  Potential Role for the BLM Helicase in Recombinational Repair via a Conserved Interaction with RAD51* , 2001, The Journal of Biological Chemistry.

[26]  F. Cross,et al.  CLB5: a novel B cyclin from budding yeast with a role in S phase. , 1992, Genes & development.

[27]  E. Louis,et al.  SGS1 is required for telomere elongation in the absence of telomerase , 2001, Current Biology.

[28]  N. Maizels,et al.  The Bloom’s Syndrome Helicase Unwinds G4 DNA* , 1998, The Journal of Biological Chemistry.

[29]  I. Hickson,et al.  RecQ family helicases: roles in cancer and aging. , 2000, Current opinion in genetics & development.

[30]  S Gangloff,et al.  The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase , 1994, Molecular and cellular biology.

[31]  I. Hickson,et al.  Replication Protein A Physically Interacts with the Bloom's Syndrome Protein and Stimulates Its Helicase Activity* , 2000, The Journal of Biological Chemistry.

[32]  R. Kolodner,et al.  Suppression of genome instability by redundant S-phase checkpoint pathways in Saccharomyces cerevisiae , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  S. Brill,et al.  Role of SGS1 and SLX4 in maintaining rDNA structure in Saccharomyces cerevisiae , 2002, Current Genetics.

[34]  K Nasmyth,et al.  CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. , 1993, Genes & development.

[35]  K. Gatter,et al.  The Bloom's Syndrome Gene Product Interacts with Topoisomerase III* , 2000, The Journal of Biological Chemistry.

[36]  A. Shimamoto,et al.  Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[37]  N. Maizels,et al.  The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs. , 1999, Nucleic acids research.

[38]  N. Kleckner,et al.  ATR Homolog Mec1 Promotes Fork Progression, Thus Averting Breaks in Replication Slow Zones , 2002, Science.

[39]  W. L. Fangman,et al.  The localization of replication origins on ARS plasmids in S. cerevisiae , 1987, Cell.

[40]  Aaron Bensimon,et al.  Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus. , 2002, Genes & development.

[41]  Judith L. Campbell,et al.  Mutations in DNA Replication Genes Reduce Yeast Life Span , 2002, Molecular and Cellular Biology.

[42]  J. Diffley,et al.  Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint , 2001, Nature.

[43]  S. Gasser,et al.  RecQ helicases: at the heart of genetic stability , 2002, FEBS letters.

[44]  Robert E. Johnson,et al.  Requirement of yeast SGS1 and SRS2 genes for replication and transcription. , 1999, Science.

[45]  J. Wang,et al.  Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S. cerevisiae. , 1999, Journal of molecular biology.

[46]  J. German,et al.  An abnormal profile of DNA replication intermediates in Bloom's syndrome. , 1990, Cancer research.

[47]  J. Diffley,et al.  A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication , 1998, Nature.

[48]  M. McVey,et al.  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. , 2001, Genetics.

[49]  J. Huberman,et al.  University of Groningen Organization of Replication of Ribosomal DNA in Saccharomyces cerevisiae , 2017 .

[50]  Hong Yan,et al.  Evidence for a Replication Function of Ffa-1, the Xenopus Orthologue of Werner Syndrome Protein , 2001, The Journal of cell biology.

[51]  Yangzhou Wang,et al.  DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast , 2001, Molecular and Cellular Biology.

[52]  W. L. Fangman,et al.  A replication fork barrier at the 3′ end of yeast ribosomal RNA genes , 1988, Cell.

[53]  L. Guarente,et al.  Extrachromosomal rDNA Circles— A Cause of Aging in Yeast , 1997, Cell.

[54]  P. Silver,et al.  Elimination of replication block protein Fob1 extends the life span of yeast mother cells. , 1999, Molecular cell.

[55]  I. Hickson,et al.  DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders. , 2001, Human molecular genetics.

[56]  C. Newlon,et al.  DNA Replication Fork Pause Sites Dependent on Transcription , 1996, Science.

[57]  K. Shirahige,et al.  Regulation of DNA-replication origins during cell-cycle progression , 1998, Nature.

[58]  J. Diffley,et al.  Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? , 2001, Current opinion in genetics & development.

[59]  C. Newlon,et al.  The DNA replication checkpoint response stabilizes stalled replication forks , 2001, Nature.

[60]  F. Fabre,et al.  Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases , 2000, Nature Genetics.

[61]  H. Klein,et al.  Replication, recombination, and repair: going for the gold. , 2002, Molecular cell.

[62]  L. Loeb,et al.  Interactions between the Werner Syndrome Helicase and DNA Polymerase δ Specifically Facilitate Copying of Tetraplex and Hairpin Structures of the d(CGG) n Trinucleotide Repeat Sequence* , 2001, The Journal of Biological Chemistry.

[63]  F. Fabre,et al.  Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication , 2002, Proceedings of the National Academy of Sciences of the United States of America.