Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks

Abstract

Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.

FIG. 1.

Homologous recombination is induced by an I-SceI-induced DSB, thymidine, and camptothecin. (A) Structure of the SCneo recombination substrate containing two nonfunctional copies of the neo^R gene. (B) Upon homologous recombination, a functional neo^R gene can be produced by short tract gene conversion, which results in a 4.0-kb product or a sister chromatid exchange/long-tract gene conversion, which restores a neo^R gene within a 7.3-kb product (16). (C) Homologous recombination frequencies (as measured by formation of G418-resistant clones) in S8SN.11 cells following transient transfection with pCMV3xnlsI-SceI or treatment with thymidine (10 mM) or camptothecin (100 nM) for 24-hour). The mean recombination frequency and standard deviation of at least three experiments are shown. Two and three stars designate statistical significance versus the control in the t test: P < 0.01 and P < 0.001, respectively.

FIG. 2.

DSBs and homologous recombination induced by camptothecin depend on replication. (A) Pulsed-field gel electrophoresis showing DNA fragmentation following a 6-hour treatment with camptothecin (200 nM) and/or aphidicolin (3 μM). (B) Homologous recombination measured as the frequency of reversion to HAsT-resistant colonies in S8SN.11 cells following a 6-hour treatment with camptothecin (200 nM) and/or aphidicolin (3 μM). The average (column) and standard deviation (error bars) of three experiments are depicted.

FIG. 3.

(A) Southern blot identifying the presence of the 4.0-kb product or 7.3-kb product in isolated G418-resistant clones. (B) Relative frequency of each recombinant product formed in approximately 40 clones isolated following either control, I-SceI, thymidine (10 mM), or camptothecin (100 nM) treatments. Numbers above the bars indicate the number of clones found in each category. (C) Number of recombination events triggered following control, I-SceI, thymidine (10 mM), or camptothecin (100 nM) treatments.

FIG. 4.

Cell cycle analysis of S8SN.11 cells following treatment with I-SceI or after a 24-hour treatment with thymidine (10 mM) or camptothecin (100 nM). A 30-minute bromodeoxyuridine (BrdU) treatment indicates replicating cells. DNA content is measured through propidium iodine staining.

FIG. 5.

SSB repair-deficient EM9 cells (XRCC1 mutated) accumulate more DSBs following camptothecin treatment. Pulsed-field gel electrophoresis of XRCC1-deficient (EM9) and wild-type (AA8) cells following a 24-hour treatment with increasing doses of camptothecin.

FIG. 6.

Elevated levels of spontaneous and camptothecin-induced γH2Ax foci and RAD51 foci in SSB repair-deficient EM9 cells. (A) Visualization of γH2Ax foci (green) and DNA (blue) in control and treated AA8 and EM9 cells. (B) Visualization of RAD51 foci (red) and DNA (blue) in control and treated AA8 and EM9 cells. (C) Percentage of cells with >5 γH2Ax foci. (D) Percentage of cells with >10 RAD51 foci. The average (column) and standard deviation (error bars) of at least three experiments are depicted.

FIG. 7.

Clones carrying both the 7.3-kb and 4-kb recombination products are mosaic and have normal recombination levels. (A) Southern blot of DNA isolated from the G418-resistant S8SN11contr.3 cell line and subclones thereof. (B) Homologous recombination frequency in the hprt gene of the G418-resistant S8SN.11 clones, carrying both the 7.3-kb and 4-kb recombination products. The average (column) and standard deviation (error bars) of at least three experiments are depicted.

FIG. 8.

Pathways for homologous recombination in mammalian cells. (A) Homologous recombination repair of a two-end DSB with synthesis-dependent strand annealing results in gene conversion with no crossover (11, 29). (B) A stalled replication fork may reverse and form a half-chicken foot structure with one protruding end. This structure is efficiently cleaved by the Mus81/Eme1 complex (6, 7) and results in a collapsed fork, which is repaired with break-induced replication (see below). Full reversal of the replication fork leads to a chicken foot structure that has one free 3′ end that is able to initiate homologous recombination to restore replication. Resolution of double Holliday junctions with topoisomerase III/BLM leads to gene conversion with no crossover (54). (C) A persisting SSB causes a collapsed replication fork which contains a one-ended DSB; this is repaired by break-induced replication (10). This collapsed replication fork is likely to be a common spontaneous substrate for homologous recombination and therefore for causing a sister chromatid exchange. For a more detailed description of the above pathways see reference . Dotted lines represent newly synthesized strands.