Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse

Abstract

Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1؊/؊ DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1؊/؊ cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM ؊/؊ cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1 ؊/؊ cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.DNA damage is continuously generated by cellular metabolic products as well as by environmental factors (23) and poses a serious threat to the cells. In cycling cells, it may inhibit the progression of replication forks, resulting in gaps and occasionally double-strand breaks (DSBs) in the daughter strands (4, 34). To release replication blocks and thereby prevent DSBs, cells have evolved two major repair pathways, translesion DNA synthesis and homologous recombination (HR) (15). Translesion synthesis is carried out by a number of specialized translesion synthesis DNA polymerases, which are able to synthesize directly across damaged DNA bases. As an alternative to translesion synthesis, bypass can be effected by a form of HR involving a transient template switch from damaged strand to the newly synthesized daughter strand on the sister chromatid (16). HR is also required for repair of DSBs arising at collapsed replication forks, caused by DNA-damaging agents such as the DNA topoisomerase I inhibitor camptothecin (1,14). This chemotherapeutic agent covalently attaches to topoisomerase I, allowing it to cleave but not religate DNA. Upon encounter with a replication fork, the topoisomerase I-cleaved DNA complex induces a single-end break (4,8,37). Restart of replication from these single-end breaks requires HR with the intact sister chromatid.The initial step of recombination-mediated DSB repair involves processing of the DSBs to produce a 3Ј single-strand overhang, followed by polymerization of Rad51 on the singlestranded DNA. In concert with Rad54 (2, 31, 38), the resulting nucleoprotein filament facilitates the homology search and pairing with the intact duplex DNA donor to form a D-loop structure. Following DNA synthesis from the invading 3Ј end, ...

Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway

“…1D and Table S1). The latter type of break is likely caused by HR-dependent repair between the broken and the intact sister chromatids, followed by defective resolution of recombination intermediates (33). Taking roles of SLX1 (21,26), XPF-ERCC1 (34)(35)(36), and MUS81-EME1 (37,38) in HR into account, our findings thus support the hypothesis that SLX4 deficiency causes impaired resolution of HR intermediates, possibly through the disruption of functional SLX4 complex, including these structure-specific endonucleases.…”

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

“…The suppressor gene was identified as the F-box helicase Fbh1 (Osman et al 2005), which has also been found in vertebrates, but not in budding yeast ( J. Kohzaki et al 2007). …”

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

“…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). This raises the question as to whether attenuation of HR by disruption of the Rad51 presynaptic filament represents a significant mechanism for HR regulation in higher eukaryotes.…”