Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Abstract

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.

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.

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.

Figure 3

The dependency on Rhp51 and Rad22 for the recombination induced by replication fork blockage at RTS1. (A) Bar chart showing the frequency of Ade⁺ recombinants in wild-type, rhp51Δ, rad22Δ and rhp51Δ rad22Δ strains containing RTS1 at site A in either orientation 1 or 2 as indicated. In order of presentation, the strains are MCW1262, MCW1433, MCW1691, MCW1692, MCW1687, MCW1688, MCW1695 and MCW1696. (B) Bar chart showing the percentage of Ade⁺ recombinants in ‘A' that are conversion-types. (C) 2-D gel analysis of replication intermediates detected by probe A in the EcoNI DNA fragment shown in Figure 2A. The strains are MCW1692 (rhp51Δ), MCW1688 (rad22Δ) and MCW1696 (rhp51Δ rad22Δ).

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).

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). (C) The same as ‘B' except that probe A was used instead of probe B.

Figure 6

The detection of one-sided DSBs in a rqh1Δ mutant containing RTS1 at site B in orientation 2. (A) Schematic of the recombination substrate showing the position of Bsu36I and EcoRV sites, and DNA probes. The region between 5.1–5.3 kb from the EcoRV site, in which the DSB maps, is also marked. (B) Analysis of genomic DNA from the rqh1Δ mutant strains MCW1438 (lane b) and MCW1445 (lane c). Genomic DNA was prepared in agarose plugs and restricted with Bsu36I and EcoRV before analysis on a 1-D gel using radiolabelled probe C. (C) The same as ‘B' except probe D was used instead of probe C. (D) 1-D gel analysis of genomic DNA from wild-type (MCW1260) and rqh1Δ (MCW1438) strains carrying plasmids as indicated. Strains were grown in EMM without thiamine for ∼20 h to allow expression of RusA from the nmt1 promoter in pREP41. RusA and RusAD70N carry C-terminal green fluorescent protein tags so their expression in the wild-type and rqh1Δ strains was confirmed by fluorescent microscopy prior to harvesting the cells for the preparation of genomic DNA. The amounts of the ∼5.3 kb band in lanes e–g relative to the total probed DNA are 2.2% (lane e), 4.5% (lane f) and 3.6% (lane g). Repetitions of this experiment show that these differences in the amount of the ∼5.3 kb band are not significant. (E) The same as ‘D' except probe D was used instead of probe C.

Figure 7

Hypothetical model for the formation of Ade⁺ recombinants following replication fork blockage at RTS1. Red boxes are the ade6 genes. The open circles indicate the position of point mutations within the ade6 genes. Further details are in the main text.