Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

Abstract

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.

Fig. 1. Recombination reporter substrate SCneo. (A) Schematic representation of SCneo and predicted neo⁺ and neo^– DSB repair products after I-SceI expression. Shaded boxes represent the 0.7 kb neo gene repeats and the open box the promoter of the S2neo gene. The small black box is the 18 bp I-SceI site. During a short tract gene conversion (STGC), the I-SceI site is replaced by the NcoI site, but SCneo is not otherwise altered. Both long tract gene conversion and unequal sister chromatid exchange (LTGC/SCE) results in expansion of the SCneo locus. The expansion typically results in the middle neo repeat containing NcoI and SalI sites. Other LTGC products are also possible, depending on the length of the conversion tract. In a non-homologous end joining (NHEJ) event, the I-SceI site is disrupted, as these events usually involve small deletions or insertions. The 0.7 kb product can arise either from a homologous deletion (HD) or as the reciprocal contraction product of an unequal SCE. HD events arise from annealing of single strands at the two neo repeats, such that one repeat and the intervening hyg gene is deleted. The relevant restriction enzyme sites are shown: H, HindIII; N, NcoI; S, SalI; B, BamHI; X, XhoI. (B) Predicted products from two distinct mechanisms of unequal sister chromatid recombination. In an LTGC, the donor of information will remain unaltered during conversion of sequences downstream of the I-SceI site. In an SCE, expansion of one chromatid to a triplication will be associated with contraction on the other chromatid.

Fig. 2. Sister chromatid recombination within the SCneo locus is not associated with reciprocal exchange. After I-SceI expression in cell lines containing the SCneo substrate, single cells were grown in non-selective medium and then replica plated to test for resistance to G418. Southern blot analysis was performed on clones containing G418^R cells using XhoI and HindIII digestion of genomic DNA. The probe was a 1.2 kb BamHI–XhoI DNA fragment containing the complete neo sequence (Figure 1A). (A) Parental V79 (4-18) cells and clones in which G418^R cells were identified. STGC events, lanes 1–4, 6, 8, 9, 12, 14 and 15; LTGC events, lanes 5, 7, 10, 11 and 13. (B) Parental V79 (4-18) cells and clones in which cells were identified to have undergone an LTGC event (lanes 1–12) or an HD event (lane 13). In both (A) and (B), each of the expansion products is presumed to be derived from an LTGC event, as none of the clones contains an associated 0.7 kb reciprocal SCE product. M, marker derived from V79 (4-18) genomic DNA digested with XhoI, BamHI and HindIII.

Fig. 3. Segregation of donor and recipient sequences after sister chromatid recombination. A clone containing 7.3 and 4.0 kb XhoI–HindIII SCneo fragments was subcloned. Each subclone contains either the 4.0 or the 7.3 kb XhoI–HindIII fragment as expected for segregation of sister chromatids to daughter cells. Presumably the 4.0 kb fragment served as the donor of information during DSB repair by LTGC to generate the 7.3 kb fragment on the recipient chromatid. Genomic DNA from each subclone was digested with XhoI and HindIII and subjected to Southern blot analysis using a neo gene probe.

Fig. 4. Structure of sister chromatid recombination products. To derive the structures, genomic DNA from each LTGC clone was digested with the indicated restriction enzymes and subjected to Southern blot analysis. The total number of events obtained for each class is indicated. H, HindIII; N, NcoI; S, SalI; B, BamHI; Bst, BstXI; Sac, SacI; Spe, SpeI; X, XhoI, (X,H), either a XhoI or HindIII site.

Fig. 5. Model for the generation of sister chromatid gene conversion events. LTGC and LTGC/NHEJ events can be initiated by strand invasion of the broken S2neo gene into the 3′ neo gene on the sister chromatid, with repair synthesis primed by the invading end. (A) In an LTGC event, repair synthesis continues into the downstream S2neo gene and is followed by homologous annealing of the newly synthesized strand to the end of the broken chromatid. (B) In an LTGC/NHEJ event, repair synthesis terminates downstream of the neo⁺ gene and is followed by NHEJ of the newly synthesized strand to the end of the broken chromatid. See text for details.