Role of the Schizosaccharomyces pombe F-Box DNA Helicase in Processing Recombination Intermediates

Abstract

In an effort to identify novel genes involved in recombination repair, we isolated fission yeast Schizosaccharomyces pombe mutants sensitive to methyl methanesulfonate (MMS) and a synthetic lethal with rad2. A gene that complements such mutations was isolated from the S. pombe genomic library, and subsequent analysis identified it as the fbh1 gene encoding the F-box DNA helicase, which is conserved in mammals but not conserved in Saccharomyces cerevisiae. An fbh1 deletion mutant is moderately sensitive to UV, MMS, and ␥ rays. The rhp51 (RAD51 ortholog) mutation is epistatic to fbh1. fbh1 is essential for viability in stationary-phase cells and in the absence of either Srs2 or Rqh1 DNA helicase. In each case, lethality is suppressed by deletion of the recombination gene rhp57. These results suggested that fbh1 acts downstream of rhp51 and rhp57. Following UV irradiation or entry into the stationary phase, nuclear chromosomal domains of the fbh1⌬ mutant shrank, and accumulation of some recombination intermediates was suggested by pulsed-field gel electrophoresis. Focus formation of Fbh1 protein was induced by treatment that damages DNA. Thus, the F-box DNA helicase appears to process toxic recombination intermediates, the formation of which is dependent on the function of Rhp51.Homologous recombination not only shuffles genetic information upon sexual reproduction but also repairs damaged DNA by use of the homologous information. Furthermore, it can regenerate replication forks when they become stalled or collapsed.Molecular mechanisms of homologous recombination in eukaryotes have been most extensively studied in the budding yeast Saccharomyces cerevisiae (reviewed in references3, 27, 42, 46, and 48). In this yeast, the MRX (Mre11 Rad50 Xrs2) complex is required for the processing of double-strand break ends to generate 3Ј-protruding ends. The resulting singlestrand regions are coated by single-strand-binding protein RPA (replication protein A). Rad52 stimulates loading of Rad51 on RPA-coated single-strand DNA to form Rad51 nucleoprotein filament. A complex of Rad55 and Rad57, which are Rad51 paralogs, is also implicated in the assembly and stabilization of Rad51 nucleoprotein filament. Rad51 nucleoprotein filament searches homologous sequences and catalyzes the exchange of strands to form a heteroduplex joint called a D loop. Rad54 facilitates D-loop formation by remodeling chromatin structures. The annealed 3Ј ends are then used as primers for repair DNA synthesis. The resulting junction molecules are resolved either by dissociation of the crossed strands or by cutting of the junction point. The Rad52 group proteins (Rad50, Rad51, Rad52, Rad54, Rad55, Rad57, Mre11, and Xrs2) are conserved throughout eukaryotes, indicating a conservation of the molecular mechanisms pertaining to homologous recombination.In addition to the aforementioned recombination factors,

The Rad52 Homologs Rad22 and Rti1 ofSchizosaccharomyces pombeAre Not Essential for Meiotic Interhomolog Recombination, but Are Required for Meiotic Intrachromosomal Recombination and Mating-Type-Related DNA Repair

“…As expected, the deletion of fbh1 in a rad22D unsuppressed background restored damage resistance to wild-type levels. Interestingly, the fbh1D mutant also showed sensitivity to DNA-damaging agents, but only to higher doses (data not shown; see Morishita et al 2005;Osman et al 2005). These previous studies demonstrated that deletion of fbh1 impaired growth and viability, probably due to chromosome segregation defects.…”

RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments

“…The mechanistic basis of the Srs2 function was elucidated by biochemical studies that revealed its ability to bind Rad51 and to dismantle the Rad51-ssDNA (single-stranded DNA) nucleoprotein filament, the key catalytic intermediate in recombination reactions (Krejci et al 2003;Veaute et al 2003;Sung and Klein 2006). To date, no apparent Srs2 ortholog has been identified in other eukaryotes, although the recently identified Fbh1 helicase shows some structural similarity to the Srs2 helicase family and studies in Schizosaccharomyces pombe suggest that Fbh1 plays a role in processing HR intermediates (Osman et al 2005;Morishita et al 2005). However, Fbh1-deficient DT40 cells show no prominent sensitivity to DNA damaging agents, and exhibit only a mild SCE phenotype (Kohzaki et al 2007).…”

FBH1 influences DNA replication fork stability and homologous recombination through ubiquitylation of RAD51

“…Previous data derived from studies in S. pombe have suggested that both the F-box domain and the helicase domain of Fbh1 are required for the repair of DNA damage in that organism, although the helicase function seems more critical 18,19 . Moreover, genetic analysis suggested that yeast Fbh1 acts downstream of Rad51 in the HR pathway 18,20 . Consistent with this, the level of Rad51 nuclear foci is greatly increased in Fbh1-deficient yeast, a phenotype that is also characteristic of FBH1-depleted human cells 15,21,22 .…”