Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase - PubMed (original) (raw)

Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase

C L Doe et al. EMBO J. 2000.

Abstract

A key stage during homologous recombination is the processing of the Holliday junction, which determines the outcome of the recombination reaction. To dissect the pathways of Holliday junction processing in a eukaryote, we have targeted an Escherichia coli Holliday junction resolvase to the nuclei of fission yeast recombination-deficient mutants and analysed their phenotypes. The resolvase partially complements the UV and hydroxyurea hypersensitivity and associated aberrant mitoses of an rqh1(-) mutant. Rqh1 is a member of the RecQ subfamily of DNA helicases that control recombination particularly during S-phase. Significantly, overexpression of the resolvase in wild-type cells partly mimics the loss of viability, hyper-recombination and 'cut' phenotype of an rqh1(-) mutant. These results indicate that Holliday junctions form in wild-type cells that are normally removed in a non-recombinogenic way, possibly by Rqh1 catalysing their reverse branch migration. We propose that in the absence of Rqh1, replication fork arrest results in the accumulation of Holliday junctions, which can either impede sister chromatid segregation or lead to the formation of recombinants through Holliday junction resolution.

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Figures

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Fig. 1. Construction of an HJ resolvase that is targeted to the eukaryotic nucleus. (A) Schematic of pREP-rus plasmid. (B) Effect of pNLS-RusA-GFP on survival of a _ruv_– _rus_– E.coli strain following UV irradiation. Survival is compared with wild-type and _ruv_– _rus_– strains transformed with the empty expression vector pT7-7. (C–F) Phase contrast and fluorescence microscopy images of a wild-type S.pombe cell transformed with pREP1-rus, grown in the absence of thiamine, and stained with DAPI. (C) Phase contrast image. (D) DAPI fluorescent image. (E) GFP image. (F) Merged image of (D) and (E).

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Fig. 2. Effect of NLS–RusA–GFP on the HU sensitivity of an _S.pombe rqh1_– strain. (A–G) Wild-type (A–C) and _rqh1_– (D–G) strains, transformed with plasmids as indicated, were cultured in the presence (D and E) or absence (A–C and F–G) of thiamine, serially diluted and spotted onto appropriately supplemented EMM plates containing the indicated amounts of HU. In each case, the neat spot (10–0) represents 105 cells plated. The strains used were MCW45 and MCW7.

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Fig. 3. Effect of NLS–RusA–GFP on UV-irradiated wild-type and _rqh1_– cells. Wild-type and _rqh1_– cells, transformed with plasmids and cultured in the presence or absence of thiamine as indicated, were assayed for colony formation before and after UV irradiation. The fraction surviving is expressed relative to unirradiated cells.

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Fig. 4. Effect of NLS–RusA–GFP on the ‘cut’ phenotype of _rqh1_– cells. (A) Examples of _rqh1_– cells undergoing normal cell division (in the absence of HU) and aberrant divisions (after exposure to HU). Images are of DAPI-stained cells viewed by fluorescence microscopy. DAPI stains nuclear DNA brightly. It also stains the rest of the cytoplasm weakly, allowing visualization of septa as dark lines across the cells. (B) Effect of NLS–RusA–GFP on the HU sensitivity of an _rqh1_– mutant. The strains were MCW45 and MCW7 transformed with plasmids as indicated. Relative viability was calculated by dividing the number of viable cells after the addition of HU by the number that were viable before its addition. (C) Effect of NLS–RusA–GFP on the proportion of dividing cells that display the ‘cut’ phenotype. Cells from the cultures described in (B) were scored for the ‘cut’ phenotype. See Materials and methods for further details.

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Fig. 5. A non-tandem direct repeat of heteroalleles for measuring recombination. (A) Schematic of intrachromosomal recombination substrate and recombinant products. Solid and open circles represent the ade6-L469 and ade6-M375 mutations, respectively. (B–G) Some of the possible mechanisms of deletion- and conversion-type recombinant formation. (B) DSB/gap repair involving intrachromatid recombination. If the recombination intermediate is resolved to give a non-crossover product, then this can generate an ade+ his+ conversion type. (C) Intrachromatid exchange between the direct repeats generates a single copy of the gene in the chromosome and an excised circle bearing the second copy together with the his+ gene. (D) Single strand annealing (SSA) can repair a DSB between two direct repeats by resection of the broken ends to expose two complementary single strands that anneal. Repair involves removal of the non-homologous 3′ ends. (E) Replication slippage involving detachment of a nascent strand and mispairing within repeats upon reattachment. (F) Break-induced replication (BIR). This may occur when a replication fork collapses after encountering a single strand break in the template DNA. Recombination can restore the fork by reattaching the broken end. If the broken arm of the fork is degraded, then recombination may occur between non-equivalent repeats, resulting in the formation of deletion-type recombinants. In this version of the BIR model, the reformation of the replication fork is accompanied by the formation and resolution of an HJ (not shown). In other versions, replication is envisaged to proceed via a migrating bubble or D-loop (Paques and Haber, 1999). (G) Unequal sister chromatid exchange can result in a deletion on one chromatid and a triplication on the other chromatid. Such unequal or slipped recombination can generate conversion-type recombinants if recombination intermediates are processed without crossing over.

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Fig. 6. Spontaneous and UV-induced recombination in wild-type and _rqh1_– cells, and the effect of pREP41-rus. (A) Mean frequency of deletion- and conversion-type recombinants. (B) The percentage of total recombinants that are conversion types. Error bars represent the 95% confidence limits.

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Fig. 7. The effect of high-level expression of NLS–RusA–GFP on spontaneous and UV-induced recombination in wild-type cells. (A) Mean frequency of deletion- and conversion-type recombinants. (B) The percentage of total recombinants that are conversion types. Error bars represent the 95% confidence limits.

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Fig. 8. Model for Rqh1 controlling recombination at stalled replication forks. In this version of the model, the replication fork is blocked by a UV photoproduct (solid triangle) in the leading strand template. The lagging strand polymerase uncouples from the leading strand polymerase and continues synthesis past the lesion site (Cordeiro-Stone et al., 1997). An HJ is formed either by a strand invasion reaction catalysed by a Rad51-like protein bound to the single-stranded region exposed on the leading strand template or by regression of the fork. Fork regression or strand exchange may provide room for repair enzymes to operate. Alternatively, the leading strand may bypass the lesion by using the extended lagging strand as a template (dashed line) (Higgins et al., 1976). Reverse branch migration of the HJ would re-establish the replication fork, which could continue synthesis unimpeded. Resolution of the HJ collapses the replication fork and promotes BIR.

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