Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast
2005; Springer Nature; Volume: 24; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7600670
ISSN1460-2075
AutoresJong Sook Ahn, Fekret Osman, Matthew C. Whitby,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle5 May 2005free access Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast Jong Sook Ahn Jong Sook Ahn Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Fekret Osman Fekret Osman Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Matthew C Whitby Corresponding Author Matthew C Whitby Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Jong Sook Ahn Jong Sook Ahn Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Fekret Osman Fekret Osman Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Matthew C Whitby Corresponding Author Matthew C Whitby Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Author Information Jong Sook Ahn1, Fekret Osman1 and Matthew C Whitby 1 1Department of Biochemistry, University of Oxford, Oxford, UK *Corresponding author. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 1865 275192; Fax: +44 1865 275297; E-mail: [email protected] The EMBO Journal (2005)24:2011-2023https://doi.org/10.1038/sj.emboj.7600670 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events. Introduction The path of a replication fork is often littered with obstacles that prevent its easy passage. Such obstacles can be accidental, for example lesions in the DNA template or proteins bound to the DNA template, or can be programmed (Rothstein et al, 2000). The appropriate response to these obstacles is important for both efficient DNA synthesis and the maintenance of genome stability. Proteins that promote homologous recombination play important roles in processing stalled and blocked replication forks (McGlynn and Lloyd, 2002; Michel et al, 2004). For example, they can promote fork reversal where the nascent strands are unwound from their templates and annealed to each other (McGlynn and Lloyd, 2002). Fork reversal may enable polymerases to switch templates and bypass DNA lesions (Higgins et al, 1976), as well as provide room for DNA repair enzymes to remove blocking lesions in the DNA template (Courcelle et al, 2003). Recombination proteins can also protect the replication fork from excessive nucleolytic attack (Courcelle et al, 2003), and help the restart of replication by generating DNA structures at which replication proteins can reload (Liu et al, 1999). However, the cell has to be circumspect about its use of recombination. Inappropriate recombination can result in genome instability, for example recombination between repetitive DNA elements can result in the expansion and contraction of tandem arrays, or even translocations between different chromosomes. Such events are hallmarks of a number of diseases including cancer. Studies of replication fork stalling in Escherichia coli have established that the way in which a blocked replication fork is processed depends on the type of block (Michel et al, 2004). For example, an ultra violet light (UV)-induced lesion in the leading strand template may be side-stepped by a recombination-mediated transient switch of the polymerase onto the nascent lagging strand (Higgins et al, 1976), whereas a UV-induced lesion in the lagging strand template may be left unreplicated, the resultant daughter strand gap being repaired by recombination postreplication (Rupp and Howard-Flanders, 1968). At other types of stalled or blocked replication fork, it may be sufficient to simply stabilise the fork until the block is removed and DNA synthesis can be resumed. This appears to be true in yeast for forks that are stalled by dNTP depletion mediated by the drug hydroxyurea (HU), which inhibits ribonucleotide reductase. The maintenance of fork stability here depends on the intra-S checkpoint kinase Rad53 (Lopes et al, 2001; Sogo et al, 2002). Members of the RecQ family of DNA helicases, which includes Sgs1 and Rqh1 in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively, and BLM and WRN in humans, also play crucial roles in maintaining fork stability (Hickson, 2003). The importance of this is underlined by the fact that defects in BLM and WRN give rise to the cancer-prone diseases Blooms syndrome and Werners syndrome, respectively. In S. cerevisiae, Sgs1 prevents polymerase dissociation from replication forks stalled by HU treatment (Cobb et al, 2003). RecQ helicases may also reset reversed replication forks by branch migrating Holliday junctions (HJs) that are formed when nascent strands anneal to each other (Doe et al, 2000; Karow et al, 2000). It has been suggested that this activity might prevent replication fork breakage by the XPF-related endonuclease Mus81-Eme1 (Kaliraman et al, 2001; Doe et al, 2002). The ability to branch migrate HJs may also be used by RecQ helicases to abort inappropriate recombination at stalled forks, and, together with Topoisomerase III, to process double HJ intermediates as noncrossover recombinants (Ira et al, 2003; Wu and Hickson, 2003). These activities would reduce the risk of genome rearrangements caused by crossing over between repeated DNA sequences. A growing number of programmed replication fork barriers (RFBs) or pause sites have been identified (Rothstein et al, 2000). Important roles for RFBs include ensuring the successful merging of opposing replication forks, and promoting the avoidance of collisions between replication forks and transcription complexes (Brewer and Fangman, 1988; Krabbe et al, 1997; Rothstein et al, 2000). Unlike most accidental blockades, the majority of programmed RFBs are unidirectional. Replication can therefore be completed when the blocked fork merges with the opposing fork. In theory, recombination at such blocked forks would be an unnecessary risk, and therefore should be suppressed. However, like accidental blockades, programmed RFBs can elicit different responses. In E. coli, replication forks blocked at Ter sites bound by the Tus protein remain stable, and only provoke recombination if a subsequent round of replication runs into the back of them to generate free double-strand ends (Bidnenko et al, 2002). In contrast, forks blocked at RFBs in the rDNA locus of S. cerevisiae appear to more directly stimulate recombination resulting in the expansion and contraction of the array, as well as the production of extrachromosomal rDNA circles (ERCs) (Kobayashi et al, 1998; Weitao et al, 2003b; Burkhalter and Sogo, 2004). Here, we have analysed whether a third RFB, RTS1, provokes recombination in S. pombe. RTS1 ensures that replication of the mat1 locus in S. pombe occurs in the centromere-proximal direction (Dalgaard and Klar, 2001; Vengrova et al, 2002). This is necessary for laying down an imprint, which stimulates mating-type switching in the next generation (Dalgaard and Klar, 1999). Essential to RTS1 function are four repeated ∼55 bp motifs, whose function is enhanced by an ∼60 bp purine-rich region (Codlin and Dalgaard, 2003). The barrier function of these cis-acting elements depends on the Swi1–Swi3 complex and the trans-acting replication termination factors Rtf1 and Rtf2 (Dalgaard and Klar, 2000). We show that replication fork blockage by RTS1 massively stimulates recombination between repeated DNA sequences with a bias toward conservative gene conversion events. This recombination, like that induced by stochastic events, depends on the S. pombe homologues of the recombinases Rad51 (called Rhp51) and Rad52 (called Rad22). These data provide the first demonstration that a site-specific replication fork blockage outside of the rDNA locus directly stimulates recombination. We also show that RTS1-induced recombination is constrained by Rqh1. In particular, rqh1− mutants exhibit a dramatic increase in deletion recombinants, which correlates with the detection of one-sided double-strand breaks (DSBs) in the vicinity of the RFB. This provides further evidence that RecQ DNA helicases protect stalled replication forks from collapse. Results Agents that stimulate direct repeat recombination To measure inter/intra-chromosomal recombination in S. pombe, we have used a direct repeat of ade6− heteroalleles with an intervening his3+ gene positioned at the ade6 locus on chromosome 3. With this system, ade6+ recombinants can be divided into those that retain the intervening marker (conversion-types) and those that lose it (deletion-types) (Figure 1A). Under our normal assay conditions, approximately 3.0–4.5 Ade+ recombinants arise spontaneously in every 104 viable cells, and typically between 70 and 80% of these are deletion-types (Figure 1B and Table I). Various agents, which are known to impede replication forks, stimulate direct repeat recombination (Galli and Schiestl, 1999). However, a comparison of their affect on the ratio of deletion and conversion-types has not been made. UV, which generates lesions such as pyrimidine dimers that block the normal replicative polymerases, stimulates the formation of conversion-types more than it does deletion-types, altering the ratio of these recombinants from approximately 1:3 to 1:1 (Figure 1B) (Osman et al, 2000). In contrast camptothecin (CPT), which inhibits the religation step during the reaction cycle of topoisomerase I and consequently generates single-strand breaks in DNA at which replication forks collapse (Pommier et al, 2003), stimulates mostly deletion-type recombinants (Figure 1C). Finally, fork stalling induced by HU only slightly stimulates deletion-type recombinants (Figure 1C). These data confirm that the impedance of a replication fork can induce recombination, and that the nature of the impedance influences the type of recombinant that is generated. Intriguingly, the fact that recombinant formation is only weakly stimulated by acute HU exposure, suggests that replication fork stalling per se is insufficient to promote recombination. Figure 1.Induction of direct repeat recombination by different genotoxins. (A) Schematic of intrachromosomal recombination substrate and recombinant products. Solid and open circles represent the ade6-L469 and ade6-M375 mutations, respectively. (B) The effect of increasing doses of UV on the frequency and type of Ade+ recombinants in a wild-type strain (MCW39). (C) The effect of a timed exposure to CPT or HU on the frequency and type of Ade+ recombinants in a wild-type strain (MCW39). Note that the ∼2-fold increase in spontaneous Ade+ recombinants in these assays compared to that in 'B' is due to the slightly different protocols that were used. Download figure Download PowerPoint Table 1. RTS1-induced mitotic recombination frequencies Strain RTS1 Mean frequency of Ade+ recombinants per 104 viable cells % Conversion typesa Site Orientation Total (His−+His+)a Deletion type (His−)a Conversion type (His+)a Wild type (MCW39) No RTS1 4.42 (±1.6) 3.47 (±1.3) 0.95 (±0.51) 21.4 (±8.0) Wild type (MCW1262) A 1 3.26 (±1.0) 2.18 (±0.76) 1.08 (±0.49) 33.6 (±11.0) Wild type (MCW1433) A 2 189.2 (±114) 86.2 (±60.1) 103.0 (±61.9) 55.9 (±10.9) Wild type (MCW1256) B 1 6.57 (±4.63) 5.75 (±4.36) 0.82 (±0.51) 13.4 (±7.2) Wild type (MCW1260) B 2 36.1 (±14.1) 24.2 (±11.2) 11.9 (±4.5) 35.1 (±11.6) swi1Δ (MCW1362) A 1 33.7 (±18.7) 25.1 (±14.8) 8.6 (±6.8) 25.2 (±12.0) swi1Δ (MCW1358) A 2 47.9 (±27.4) 36.2 (±24.2) 11.7 (±7.5) 27.0 (±11.0) swi1Δ (MCW1395) B 1 37.6 (±21.7) 30.8 (±19.0) 6.77 (±4.15) 19.1 (±9.4) swi1Δ (MCW1377) B 2 20.9 (±13.1) 16.8 (±10.9) 4.1 (±3.3) 19.3 (±9.2) rhp51Δ (MCW1691) A 1 10.72 (±5.6) 10.7 (±5.6) 0.02 (±0.04) 0.19 (±0.45) rhp51Δ (MCW1692) A 2 34.2 (±17.3) 32.6 (±16.7) 1.55 (±1.06) 4.71 (±3.4) rad22Δ (MCW1687) A 1 1.02 (±0.49) 1.0 (±0.48) 0.016 (±0.037) 1.2 (±2.7) rad22Δ (MCW1688) A 2 3.39 (±1.6) 3.27 (±1.59) 0.12 (±0.17) 3.57 (±4.52) rhp51Δ rad22Δ (MCW1695) A 1 1.09 (±0.52) 1.09 (±0.52) 0.005 (±0.02) 0.33 (±1.3) rhp51Δ rad22Δ (MCW1696) A 2 2.52 (±1.33) 2.49 (±1.36) 0.028 (±0.057) 1.75 (±3.36) rqh1Δ (MCW1443) A 1 11.95 (±3.9) 10.2 (±3.4) 1.75 (±0.77) 15.1 (±5.7) rqh1Δ (MCW1447) A 2 2410 (±1579) 2152 (±1530) 258 (±179) 13.1 (±12.1) rqh1Δ (MCW1445) B 1 17.4 (±8.9) 14.6 (±6.0) 2.8 (±4.6) 12.8 (±10.7) rqh1Δ (MCW1438) B 2 748.7 (±266) 678.8 (±243) 69.9 (±43.4) 9.2 (±4.7) a The values in parentheses are the standard deviations. The RTS1 RFB stimulates recombination when placed at the ade6 locus To study the effect of a site-specific RFB on direct repeat recombination, we have engineered strains in which an 889 bp fragment containing the RTS1 element has been placed at two different positions within the ade6− heteroallelic repeat described above (Figure 2A). The two different positions for RTS1 are between the his3+ and ade6-M375 genes (site A), and within the ade6-L469 coding region (site B). As RTS1 is a polar RFB, strains have been constructed that contain RTS1 in both possible orientations at sites A and B. RTS1 in orientation 1 is active for blocking forks coming from the direction of the centromere (cen3), whereas orientation 2 blocks forks moving toward the centromere (Figure 2A). Although the direction of replication across the ade6 locus is unknown, the positions of putative origins of replication are such that there is a predicted bias in replication proceeding across the ade6 locus towards cen3 (Figure 2A) (Segurado et al, 2003). If true then only RTS1 in orientation 2 would result in replication forks being blocked within the ade6− direct repeat. To investigate this, we used a native two-dimensional (2-D) agarose gel electrophoresis technique for studying replication intermediates (Brewer and Fangman, 1987). Genomic DNA from logarithmically growing cells was digested with EcoNI, enriched for replication intermediates by fractionation on BND-cellulose, and separated in a 2-D gel. The gel was then analysed by Southern blotting using radiolabelled probe A, which is specific to the his3+ gene (Figure 2A). An uninterrupted arc of Y-shaped replication forks was detected in a strain without the RTS1 element and in one with RTS1 at site A in orientation 1 (Figure 2B). However, in the strain with RTS1 at site A in orientation 2 the replication forks accumulate as a comet-shaped spot at the top of the ascending part of the Y-arc (Figure 2B). Closer inspection of this comet-shaped signal reveals that it consists of at least two discrete spots (Figure 2B, inset). This is consistent with replication fork blockage by RTS1, which has previously been shown to consist of four repeated motifs that may each act as a barrier (Codlin and Dalgaard, 2003). These data are also consistent with the predicted bias in the direction of replication across the ade6− repeat. Figure 2.Effect of replication fork blockage by RTS1 on direct repeat recombination. (A) Schematic showing the position of the intrachromosomal recombination substrate on chromosome III. Sites of RTS1 insertion within the recombination substrate are also indicated as is probe (A) used to detect replication intermediates. (B) 2-D gel analysis of replication intermediates within the region delineated by the EcoNI restriction sites shown in 'A'. The far left panel is a guide for interpreting 2-D gels. The panels to the right of this show the 2-D gel analysis of DNA from asynchronously grown cultures of the wild-type strains MCW39 (no RTS1), MCW1262 (RTS1 site A orientation 1), and MCW1433 (RTS1 site A orientation 2). The inset picture shows an enlargement of the RFB signal with arrows indicating that it consists of at least two discrete spots. (C) Bar charts showing the effect of RTS1 at different positions in the recombination substrate on the frequency and type of Ade+ recombinants in wild-type and swi1Δ mutant strains. In order of presentation the strains are MCW1262, MCW1433, MCW1362, MCW1358, MCW1256, MCW1260, MCW1395 and MCW1377. Error bars represent the standard deviations about the mean. Download figure Download PowerPoint Having established that RTS1 acts as a strong RFB when placed within the ade6− direct repeat, we determined its effect on the frequency of recombination between the repeats (Figure 2C and Table I). In wild-type strains with RTS1 in orientation 1, either at site A or B, the frequency of Ade+ recombinants is not significantly different from that in a wild-type strain without the RTS1 element. However, strains with RTS1 at site B do produce fewer conversion-type recombinants. This is probably due to impedance of conversion-type recombination by the presence of a large chunk of heterologous sequence (the RTS1 element) within ade6-L469. In wild-type strains with RTS1 in orientation 2, the recombinant frequency increases by ∼50-fold at site A and ∼5-fold at site B compared to the equivalent strains with RTS1 in orientation 1 (Figure 2C and Table I). As replication fork blockage is specific to orientation 2 within the ade6− repeat, these data indicate that replication forks blocked by RTS1 strongly provoke recombination. Intriguingly, the proportion of these induced recombinants that are conversion-types is significantly greater than in the spontaneous recombinants (P= 90% of conversion-types are ade6-M375 to ade+ irrespective of the position and orientation of RTS1. However, in the case of the strain containing RTS1 in orientation 2 at site A, 100% of conversion-types are ade6-M375 to ade+ compared to 92% for the equivalent strain with RTS1 in orientation 1. This difference is statistically significant (P=3.5 × 10−5). Similarly, the percentage of conversion-types that are ade6-L469 to ade+ is more with RTS1 at site B in orientation 2 (10%) than if it is in orientation 1 (4%). Again this difference is statistically significant (P= 12-fold over the equivalent wild-type strain, while the increase is >20-fold with RTS1 at site B. Again the percentage of these recombinants that are conversion-types is reduced significantly in a rqh1Δ mutant (Figure 4B and Table I). No difference in the growth/viability of rqh1− strains with or without RTS1 in either orientation 1 or 2 was detected (data not shown). The increased frequency of deletion-types is therefore not due to a detrimental effect on growth of RTS1 in orientation 2 in the absence of Rqh1. Instead, these data show that Rqh1 limits the recombination that is induced by the RTS1 RFB, and in particular it limits the formation of deletion-type recombinants. Figure 4.Effect of rqh1Δ mutation on the frequency and type of recombinants induced by replication fork blockage at RTS1. (A) Bar chart showing the frequency of Ade+ recombinants in wild-type and rqh1Δ strains containing RTS1 at site A or B in orientation 1 or 2 as indicated. In order of presentation, the strains are MCW1262, MCW1433, MCW1443, MCW1447, MCW1256, MCW1260, MCW1445 and MCW1438. (B) Bar chart showing the percentage of Ade+ recombinants in 'A' that are conversion-types. (C) Schematic of a 2-D gel showing the expected position of the cone-shaped signal that would be indicative of fork reversal. (D) 2-D gel analysis of replication intermediates detected by probe A in the EcoNI DNA fragment from a rqh1Δ mutant containing RTS1 at site A in orientation 2 (MCW1447). Download figure Download PowerPoint Stalled/blocked replication forks can reverse, either spontaneously or by an enzyme-driven reaction, to form a double-stranded tail of nascent DNA onto which recombinases can load and promote recombination (McGlynn and Lloyd, 2002). One way in which RecQ DNA helicases are thought to limit recombination at stalled/blocked replication forks is by resetting reversed forks (Doe et al, 2000; Karow et al, 2000). Reversed forks have recently been observed by 2-D gel analysis at the replication fork pause site within mat1 of S. pombe (Vengrova and Dalgaard, 2004). These reversed forks appear as a cone-shaped signal displaced up from the Y-arc at about the position of the replication pause site (Figure 4C). This signal was observed to increase in a rqh1Δ mutant consistent with the idea that Rqh1 resets reversed forks (Vengrova and Dalgaard, 2004). To see if forks blocked at RTS1 were undergoing reversal, we performed a 2-D gel analysis of genomic DNA from a rqh1Δ mutant that contained RTS1 in orientation 2 at site A (Figure 4D). Consistent with our other analyses of DNA from strains with RTS1 in this position, a strong RFB is detected. In addition, a triangular-shaped signal emanating from the descending portion of the Y-arc can be seen. Previous studies have shown that this signal represents a mixture of double-Y- and X-shaped molecules formed by converging replication forks and, in the case of the X-shaped molecules, by recombination. However, we are unable to detect a cone-shaped signal indicative of fork reversal. Therefore, if fork reversal occurs at the RTS1 RFB, it is below the detection limits of our assay. It should be noted that previous 2-D gel analyses of the RTS1 RFB have also not detected evidence of fork reversal in wild-type cells (Dalgaard and Klar, 2001; Codlin and Dalgaard, 2003). Detection of one-sided DSBs at the RTS1 RFB in rqh1Δ strains It has been suggested that in the absence of Rqh1, stalled/blocked replication forks might be more susceptible to being cleaved by an endonuclease like Mus81-Eme1 (Kaliraman et al, 2001; Doe et al, 2002). This kind of endonucleolytic cleavage would detach one arm of the fork to produce a one-sided DSB. To see if this happens at the RTS1 RFB, genomic DNA from wild-type and rqh1Δ strains, which contain RTS1 at site A, was digested with PstI, separated on a 1-D gel, and analysed by Southern blotting using radiolabelled probes A and B (Figure 5). Genomic DNA was prepared and restricted in agarose plugs to prevent it shearing. Probes A and B are specific to regions that flank RTS1 at site A (Figure 5A). Both probes detect the full-length PstI fragment from wild-type and rqh1Δ DNA (Figure 5B and C). The full-length band is ∼9.2 kb but runs slightly slower (∼9.5 kb) than this when compared to the DNA ladder (lane e). This is probably because its migration is retarded by the agarose plug. An additional faster migrating band, which is indicative of a DSB, is detected with probe B in DNA from the rqh1Δ strain containing RTS1 in orientation 2 (i.e. the orientation in which replication fork blockage is detected) (Figure 5B, lane d). This band is ∼7.6 kb based on a comparison with the DNA ladder. However, as noted above, it is likely that the actual size of this band is a few hundred base pairs shorter. As this band is not detected by probe A (Figure 5C, lane d), the DSB site can be mapped approximately to the RTS1 element. If this was a two-sided DSB then a ∼2 kb band would be detected by probe A. However, only the full-length PstI fragment is detected with this probe (Figure 5C, lane d). These data indicate that DSBs are formed at or near RTS1 in the replicated arms of the blocked replication fork in the absence of Rqh1. Quantification of the bands in Figure 5B lane d indicates that ∼1.9% of the genomic DNA is broken in this way. Figure 5.Detection of one-sided DSBs in a rqh1Δ mutant containing RTS1 at site A in orientation 2. (A) Schematic of the recombination substrate showing the position of PstI sites and DNA probes. The region between 7 and 7.6 kb from the right-hand PstI site, in which the DSB maps, is also marked. (B) Analysis of genomic DNA, prepared in agarose plugs and restricted with PstI, on a 1-D gel using radiolabelled probe B. The strains are MCW1262 (lane a), MCW1443 (lane b), MCW1433 (lane c) and MCW1447 (lane d). (
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