Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

[1]  W. Heyer,et al.  RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae , 2008, Nucleic acids research.

[2]  Matthew J. Brauer,et al.  Slow Growth Induces Heat-shock Resistance in Normal and Respiratory-deficient Yeast , 2022 .

[3]  Stephen C. West,et al.  RTEL1 Maintains Genomic Stability by Suppressing Homologous Recombination , 2008, Cell.

[4]  M. Whitby,et al.  The FANCM Ortholog Fml1 Promotes Recombination at Stalled Replication Forks and Limits Crossing Over during DNA Double-Strand Break Repair , 2008, Molecular cell.

[5]  E. Sage,et al.  Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells. , 2008, DNA repair.

[6]  H. Shinagawa,et al.  Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair , 2008, BMC Molecular Biology.

[7]  F. Fabre,et al.  The Srs2 helicase activity is stimulated by Rad51 filaments on dsDNA: implications for crossover incidence during mitotic recombination. , 2008, Molecular cell.

[8]  Michael G. Sehorn,et al.  RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments. , 2007, Genes & development.

[9]  E. Egelman,et al.  Novel pro- and anti-recombination activities of the Bloom's syndrome helicase. , 2007, Genes & development.

[10]  M. Foiani,et al.  RecQ helicases queuing with Srs2 to disrupt Rad51 filaments and suppress recombination. , 2007, Genes & development.

[11]  A. Baryshnikova,et al.  The Human F-Box DNA Helicase FBH1 Faces Saccharomyces cerevisiae Srs2 and Postreplication Repair Pathway Roles , 2007, Molecular and Cellular Biology.

[12]  F. Hanaoka,et al.  Rad54 dissociates homologous recombination intermediates by branch migration , 2007, Nature Structural &Molecular Biology.

[13]  I. Hickson,et al.  Molecular genetics of RecQ helicase disorders , 2007, Cellular and Molecular Life Sciences.

[14]  D. Hoyle,et al.  Growth control of the eukaryote cell: a systems biology study in yeast , 2007, Journal of biology.

[15]  J. Sale,et al.  Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse , 2007, Molecular and Cellular Biology.

[16]  Robert J. D. Reid,et al.  The Role of DNA Double-Strand Breaks in Spontaneous Homologous Recombination in S. cerevisiae , 2006, PLoS genetics.

[17]  D. Sherratt,et al.  Replication fork blockage by transcription factor-DNA complexes in Escherichia coli , 2006, Nucleic acids research.

[18]  Wolf-Dietrich Heyer,et al.  Some disassembly required: role of DNA translocases in the disruption of recombination intermediates and dead-end complexes. , 2006, Genes & development.

[19]  Wolf-Dietrich Heyer,et al.  Rad54: the Swiss Army knife of homologous recombination? , 2006, Nucleic acids research.

[20]  D. J. Clarke,et al.  Genome‐wide replication profiles of S‐phase checkpoint mutants reveal fragile sites in yeast , 2006, The EMBO journal.

[21]  O. Mazina,et al.  Rad54 protein promotes branch migration of Holliday junctions , 2006, Nature.

[22]  Ian Grainge,et al.  Tracking of controlled Escherichia coli replication fork stalling and restart at repressor‐bound DNA in vivo , 2006, The EMBO journal.

[23]  A. Estreicher,et al.  Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe , 2006, Chromosoma.

[24]  P. Sung,et al.  Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation. , 2005, Biochemical Society transactions.

[25]  T. Toda,et al.  Role of the Schizosaccharomyces pombe F-Box DNA Helicase in Processing Recombination Intermediates , 2005, Molecular and Cellular Biology.

[26]  M. Whitby,et al.  The F-Box DNA Helicase Fbh1 Prevents Rhp51-Dependent Recombination without Mediator Proteins , 2005, Molecular and Cellular Biology.

[27]  Boris Pfander,et al.  SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase , 2005, Nature.

[28]  Efterpi Papouli,et al.  Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. , 2005, Molecular cell.

[29]  A. Carr,et al.  Gross Chromosomal Rearrangements and Elevated Recombination at an Inducible Site-Specific Replication Fork Barrier , 2005, Cell.

[30]  M. Whitby,et al.  Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast , 2005, The EMBO journal.

[31]  T. Petes,et al.  Chromosomal Translocations in Yeast Induced by Low Levels of DNA Polymerase A Model for Chromosome Fragile Sites , 2005, Cell.

[32]  F. Fabre,et al.  UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli , 2005, The EMBO journal.

[33]  L. Symington,et al.  Recombination proteins in yeast. , 2004, Annual review of genetics.

[34]  Frank Eisenhaber,et al.  S. pombe meiotic linear elements contain proteins related to synaptonemal complex components , 2004, Journal of Cell Science.

[35]  Jaehoon Kim,et al.  SCFhFBH1 can act as helicase and E3 ubiquitin ligase. , 2004, Nucleic acids research.

[36]  M. Whitby,et al.  The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast. , 2004, Nucleic acids research.

[37]  Anna Malkova,et al.  Srs2 and Sgs1–Top3 Suppress Crossovers during Double-Strand Break Repair in Yeast , 2003, Cell.

[38]  M. Poidevin,et al.  Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast. , 2003, Nucleic acids research.

[39]  M. Whitby,et al.  Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis. , 2003, Molecular cell.

[40]  D. Sherratt,et al.  Spatial and temporal organization of replicating Escherichia coli chromosomes , 2003, Molecular microbiology.

[41]  Ying Li,et al.  DNA helicase Srs2 disrupts the Rad51 presynaptic filament , 2003, Nature.

[42]  F. Fabre,et al.  The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments , 2003, Nature.

[43]  M. Whitby,et al.  Cleavage of Model Replication Forks by Fission Yeast Mus81-Eme1 and Budding Yeast Mus81-Mms4 , 2003, The Journal of Biological Chemistry.

[44]  J. C. Hope,et al.  The severe slow growth of Δsrs2 Δrqh1 in Schizosaccharomyces pombe is suppressed by loss of recombination and checkpoint genes , 2002 .

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

[46]  Z. Pan,et al.  The Novel Human DNA Helicase hFBH1 Is an F-box Protein* , 2002, The Journal of Biological Chemistry.

[47]  S. W. Wang,et al.  Involvement of Schizosaccharomyces pombe Srs2 in cellular responses to DNA damage. , 2001, Nucleic acids research.

[48]  M. Whitby,et al.  Partial suppression of the fission yeast rqh1− phenotype by expression of a bacterial Holliday junction resolvase , 2000, The EMBO journal.

[49]  A. Carr,et al.  Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. , 1998, Gene.

[50]  J. Bähler,et al.  Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis , 1993, The Journal of cell biology.

[51]  K. Maundrell.  Thiamine-repressible expression vectors pREP and pRIP for fission yeast. , 1993, Gene.

[52]  N. Sonenberg,et al.  Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF‐4A. , 1992, The EMBO journal.

[53]  R. Schiestl,et al.  The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. , 1990, Genetics.

[54]  R. Chanet,et al.  RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. , 1989, Nucleic acids research.

[55]  H. Klein,et al.  Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. , 1988, Genetics.

[56]  J. Huberman,et al.  The in vivo replication origin of the yeast 2μm plasmid , 1987, Cell.

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

[58]  F. E. Yoder,et al.  Chromosome fragile sites. , 1985, Cancer genetics and cytogenetics.

[59]  C. Lawrence,et al.  Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants , 1979, Journal of bacteriology.

[60]  J. Haber,et al.  Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. , 2009, Genes & development.

[61]  Matthew J. Brauer,et al.  Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. , 2008, Molecular biology of the cell.

[62]  M. Whitby,et al.  DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51. , 2004, Nucleic acids research.

[63]  A. Brazma,et al.  Global transcriptional responses of fission yeast to environmental stress. , 2003, Molecular biology of the cell.

[64]  S. Moreno,et al.  Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. , 1991, Methods in enzymology.