Helicase activity is only partially required for <i>Schizosaccharomyces pombe</i> Rqh1p function

Ahmad, Fouzia; Kaplan, Craig D.; Stewart, Elspeth

doi:10.1002/yea.917

Cited by 17 publications

(21 citation statements)

References 87 publications

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“…We had shown that suppression of ⌬top3 lethality was suppressed more efficiently when rqh1 ϩ was deleted compared with when its helicase activity was inactivated (42). Ahmad et al (43) showed that recovery from S-phase arrest by Rqh1 only partially depended on its helicase activity. In S. cerevisiae, the ability of Sgs1 to suppress crossovers and reduce GC tract lengths is reportedly independent of its helicase activity (J. Nickloff, personal communications).…”

Section: The Suppression Of Scc By Rqh1 Is Independent Of Its Helicasmentioning

confidence: 99%

Rqh1 blocks recombination between sister chromatids during double strand break repair, independent of its helicase activity

Hope

Mense

Jalakas

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Many questions remain about the process of DNA double strand break (DSB) repair by homologous recombination (HR), particularly concerning the exact function played by individual proteins and the details of specific steps in this process. Some recent studies have shown that RecQ DNA helicases have a function in HR. We studied the role of the RecQ helicase Rqh1 with HR proteins in the repair of a DSB created at a unique site within the Schizosaccharomyces pombe genome. We found that DSBs in rqh1 ؉ cells, are predominantly repaired by interchromosomal gene conversion, with HR between sister chromatids [sister-chromatid conversion (SCC)], occurring less frequently. In ⌬rqh1 cells, repair by SCC is favored, and gene conversion rates slow significantly. When we limited the potential for SCC in ⌬rqh1 cells by reducing the length of the G2 phase of the cell cycle, DSB repair continued to be predominated by SCC, whereas it was essentially eliminated in wild-type cells. These data indicate that Rqh1 acts to regulate DSB repair by blocking SCC. Interestingly, we found that this role for Rqh1 is independent of its helicase activity. In the course of these studies, we also found nonhomologous end joining to be largely faithful in S. pombe, contrary to current belief. These findings provide insight into the regulation of DSB repair by RecQ helicases.homologous recombination ͉ nonhomologous end joining ͉ rqh1 ͉ gene conversion D ouble strand breaks (DSBs) pose a major problem for genomic instability and cell survival, because a single unrepaired DSB is, presumably, sufficient to cause cell death (1). The sources of DSBs can be either endogenous, such as those induced during the reshuffling of DNA in Ig gene diversification, or exogenous, such as those induced by exposure to ionizing radiation (2-4). The cell has two major mechanisms for the repair of DSBs: homologous recombination (HR) and nonhomologous end joining (NHEJ), each used to varying degrees in different organisms (2,3,5,6). HR is characterized as an error-free process using homologous sequences as the template to repair the DSB. Repair by HR begins with the formation of 3Ј single strand ends at the break that can then invade homologous duplex DNA. The 3Ј end of the invading strand is extended by DNA polymerase. At this point, the DSB can be repaired by either DSB repair, which involves formation of a double Holliday junction (HJ) or synthesis-dependent strand annealing (3, 7).In NHEJ, the DNA ends are resealed by rejoining the broken ends; however, this process can lead to loss of information at the break and is, thus, referred to as error-prone repair (5,8). Whereas mammalian cells preferentially use NHEJ over HR, budding yeast, for the most part, use HR over NHEJ. In Saccharomyces cerevisiae, the MRX (MRN in Schizosaccharomyces pombe) complex has been shown to play a role in NHEJ (9, 10). In S. pombe, only pKu70͞80 and Ligase IV have been identified as functioning in NHEJ, and MRN does not appear to be active in this process (11).RecQ DNA helicases are found in vi...

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Section: The Suppression Of Scc By Rqh1 Is Independent Of Its Helicasmentioning

confidence: 99%

Rqh1 blocks recombination between sister chromatids during double strand break repair, independent of its helicase activity

Hope

Mense

Jalakas

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Helicase-dead Rqh1 mutants have intermediate phenotypes for DNA damage sensitivity and suppression of top3⌬ lethality compared to the deletion mutant (2,39). We recently reported that while rqh1⌬ mutants show a decreased frequency of DSB repair by GC, a helicase-dead mutant (rqh1:K547I [39]) showed near-WT frequencies of GC (29).…”

Section: Resultsmentioning

confidence: 99%

Mus81-Eme1-Dependent and -Independent Crossovers Form in Mitotic Cells during Double-Strand Break Repair in Schizosaccharomyces pombe

Hope

Delgado‐Cruzata

Duvshani

et al. 2007

Molecular and Cellular Biology

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During meiosis, double-strand breaks (DSBs) lead to crossovers, thought to arise from the resolution of double Holliday junctions (HJs) by an HJ resolvase. In Schizosaccharomyces pombe, meiotic crossovers are produced primarily through a mechanism requiring the Mus81-Eme1 endonuclease complex. Less is known about the processes that produces crossovers during the repair of DSBs in mitotic cells. We employed an inducible DSB system to determine the role of Rqh1-Top3 and Mus81-Eme1 in mitotic DSB repair and crossover formation in S. pombe. In agreement with the meiotic data, crossovers are suppressed in cells lacking Mus81-Eme1. And relative to the wild type, rqh1⌬ cells show a fourfold increase in crossover frequency. This suppression of crossover formation by Rqh1 is dependent on its helicase activity. We found that the synthetic lethality of cells lacking both Rqh1 and Eme1 is suppressed by loss of swi5 ؉ , which allowed us to show that the excess crossovers formed in an rqh1⌬ background are independent of Mus81-Eme1. This result suggests that a second process for crossover formation exists in S. pombe and is consistent with our finding that deletion of swi5 ؉ restored meiotic crossovers in eme1⌬ cells. Evidence suggesting that Rqh1 also acts downstream of Swi5 in crossover formation was uncovered in these studies. Our results suggest that during Rhp51-dependent repair of DSBs, Rqh1-Top3 suppresses crossovers in the Rhp57-dependent pathway while Mus81-Eme1 and possibly Rqh1 promote crossovers in the Swi5-dependent pathway.Meiotic cells depend on crossovers for creating genomic rearrangements, a critical step in ensuring genomic vitality. In fact crossing over is essential for spore viability in yeasts, while in mouse cells, blocking crossing over leads to embryonic lethality (6,17,35). The classic model for crossing over was first suggested by Robin Holliday, who proposed that crossovers arose by resolution of Holliday junctions (HJs) (27). This model was later refined to include the resolution of a double HJ (dHJ), which remains the major model for crossover formation (48) (Fig.

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“…Integration of rqh1 À DNA into the S. pombe genome Construction of strains rqh1-KA (ECS218) and rqh1-KR (ECS220) has been described previously (Ahmad et al 2002); however, strains ECS218 and ECS220 are themselves the result of backcrossing (the original strains correspond to ECS194 and 196, respectively). To integrate the rqh1 À truncation mutants into the S. pombe genome at the rqh1 + locus, the entire ORF, together with regions upstream and downstream of the rqh1 + gene, was excised from plasmids pECS208 and pECS213-220 using a combination of Sph I, Aat II and Sac I.…”

Section: Construction Of Rqh1 à Truncation Mutantsmentioning

confidence: 99%

“…The Escherichia coli RecQ protein (the prototype member of this family) and all other RecQ homologues tested possess ATP-dependent 3¢ to 5¢ DNA helicase activity (Umezu and Nakayama 1993;Karow et al 1997;Bennett et al 1998;Shen et al 1998;Ahmad et al 2002;Cui et al 2003). The Wrn protein also possesses an exonuclease domain at its N-terminal end (Shen et al 1998).…”

Section: Introductionmentioning

confidence: 97%

“…Cells lacking Rqh1 are hypersensitive to DNA damage and S-phase arrest, and both conditions result in high levels of recombination (Murray et al 1997;Stewart et al 1997;Ahmad et al 2002). Rqh1 has 3¢ to 5¢ DNA helicase activity, which contributes to the ability to tolerate DNA damage and survive S-phase arrest; in addition to this activity some other aspect of Rqh1 function is required for complete tolerance to such treatments (Ahmad et al 2002;Laursen et al 2003). A genetic interaction between rqh1 + and top3 + has also been demonstrated, as deletion of rqh1 + can rescue the lethality associated with top3D mutations in S. pombe (Goodwin et al 1999).…”

Section: Introductionmentioning

confidence: 99%

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The N-terminal region of the Schizosaccharomyces pombe RecQ helicase, Rqh1p, physically interacts with Topoisomerase III and is required for Rqh1p function

Ahmad

Stewart

2005

Mol Genet Genomics

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The Schizosaccharomyces pombe rqh1+ gene encodes a member of the RecQ DNA helicase family. Members of this protein family are essential for the maintenance of genetic integrity. Thus, mutations in the genes encoding the human RecQ homologues Blm, Wrn and RecQ4 cause Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively-diseases which result from genome instability. S. pombe cells that lack a functional rqh1+ gene show reduced viability and display defective chromosome segregation, particularly after UV irradiation or S-phase arrest. In this study we used an rqh1+ deletion series to show that the N-terminal portion of Rqh1 is essential for Rqh1 function. Moreover, the conserved Helicase and RNaseD C-terminal (HRDC) domain of Rqh1 also plays a role in allowing cells to tolerate exposure to DNA damaging agents and the S-phase inhibitor hydroxyurea (HU). We also demonstrate that Topoisomerase III (Top3) binds to a site within the first 322 N-terminal amino acids of Rqh1 and that this binding correlates with Rqh1 function. Genetic analysis of rqh1- top3delta mutants reveals that, in the presence of functional or partially functional Rqh1 protein, Top3 is required to maintain genome integrity and cell viability.

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Helicase activity is only partially required for Schizosaccharomyces pombe Rqh1p function

Cited by 17 publications

References 87 publications

Rqh1 blocks recombination between sister chromatids during double strand break repair, independent of its helicase activity

Rqh1 blocks recombination between sister chromatids during double strand break repair, independent of its helicase activity

Mus81-Eme1-Dependent and -Independent Crossovers Form in Mitotic Cells during Double-Strand Break Repair in Schizosaccharomyces pombe

The N-terminal region of the Schizosaccharomyces pombe RecQ helicase, Rqh1p, physically interacts with Topoisomerase III and is required for Rqh1p function

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