Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged (“homeologous”) sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally ≤100 bp, even in Msh2^−/− cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.The ability of a cell to repair DNA damage is integral to maintaining genome integrity. One common type of damage that is particularly detrimental is a double-strand break (DSB), where both strands of DNA are broken. If not accurately repaired, DSBs can lead to cell death, chromosomal rearrangements, and loss of genetic material (reviewed in references 14 and 19). One mechanism of DSB repair is homologous recombination (HR), in which an unbroken homologous sequence, the donor of genetic information, is used as a template for repair of the broken sequence, the recipient of genetic information. HR intermediates possess heteroduplex DNA (hDNA), where one strand of DNA is derived from the donor sequence, and the second strand is derived from the recipient sequence. Mismatches in hDNA are substrates of the mismatch repair machinery (MMR) (reviewed in reference 38), leading to gene conversion. HR is the preferred repair pathway of DSBs in Saccharomyces cerevisiae (reviewed in references 42 and 46), plays an important role in repair of DSBs in Drosophila (1, 32), and is a major repair pathway of DSBs that occur during S/G₂ in mammalian cells (33, 54).Two pathways appear to predominate for the repair of DSBs by HR, both of which can give rise to noncrossover products, which predominate in mitotic mammalian cells (Fig. ​(Fig.1)1) (29, 52, 60). In the DSB repair model proposed by Szostak et al. (61), double Holliday junctions are resolved to result in recombinant products (Fig. ​(Fig.1A).1A). More recent evidence suggests the existence of an alternative pathway, termed synthesis-dependent strand annealing (SDSA) (Fig. ​(Fig.1B)1B) (20, 40, 42, 52). One difference between these two pathways is that the DSB repair model requires capture of both DNA ends (Fig. ​(Fig.1A),1A), which can lead to bidirectional gene conversion tracts. In contrast, SDSA can involve only one end of the broken DNA followed by dissociation (Fig. ​(Fig.1B),1B), resulting in predominantly unidirectional gene conversion tracts. Another difference is that the donor sequence can be altered during DSB repair while it typically remains unchanged after SDSA.Open in a separate windowFIG. 1.Models for noncrossover gene conversion resulting from DSB repair. DSB repair is initiated by resection of the DNA ends (black; strand directionality is designated a 3′ “tail”). The resected 3′ overhang invades the homologous donor template (gray), forming hDNA at the site of invasion (i), which acts as a primer/template for repair synthesis (gray dotted line). (A) In the canonical DSB repair (DSBR) model, the second strand of the DSB is captured, resulting in another stretch of hDNA (ii) and repair synthesis, to form a double Holliday junction. Depending on how the double Holliday junction is cleaved (arrowheads), resolution can result in a crossover (data not shown) or a noncrossover, as shown. (B) In SDSA, the newly synthesized strand dissociates from the D-loop and anneals to the other DNA end to form another stretch of hDNA (iii). Repair synthesis and ligation result in a noncrossover product. While one-end invasion is illustrated for the SDSA model, it is possible for both DNA ends to invade, resulting in gene conversion on both sides of the DSB (data not shown). In both models, hDNA formed by the newly synthesized strands can be repaired by MMR, resulting in gene conversion of markers (data not shown).HR repair is sensitive to differences between the recombining sequences, and cells have developed ways to suppress recombination between diverged sequences. This suppression of “homeologous” recombination reduces HR both between diverged repeats and with foreign DNA. Suppression of homeologous recombination is conserved across species and requires the MMR machinery (7, 10, 11, 49, 56). For example, MSH2 dramatically reduces both gene targeting (12) and DSB-induced HR (15) between sequences with >1% divergence in murine embryonic stem (ES) cells.Another protein that has been proposed to suppress homeologous recombination is Sgs1, the budding yeast RecQ helicase, as sequence divergence has little effect on recombination frequencies in Sgs1 mutants (39, 59). Sgs1 mutants have other phenotypes as well; for example, they demonstrate a hyperrecombination phenotype associated with spontaneous repair (22, 65, 68). The mammalian homolog of Sgs1 is BLM, mutants of which also have a hyperrecombination phenotype, as evidenced by a high frequency of sister-chromatid exchange (SCE) in both human and mouse cells (18, 24, 34, 69). Evidence suggests that Drosophila BLM, like Sgs1, has a role in the suppression of homeologous recombination (30) although mammalian BLM has not been tested in this regard. Supporting a possible role for BLM in suppressing homeologous recombination is the observation that BLM associates with MMR factors in a large protein complex (64; reviewed in reference 21), and BLM directly interacts with two components of the MMR machinery, MLH1 (45) and MSH6 (44), which, like MSH2, is known to suppress homeologous recombination (13).To gain more insight into mammalian HR mechanisms, as well as factors that control recombination between homeologous sequences, we examined recombination between homologous and homeologous sequences in both murine and human cells. By taking advantage of multiple, single base pair polymorphisms distributed along the donor in gene conversion substrates, we examined both the nature of gene conversion tracts and the fate of the donor sequence. Unidirectional tracts with a bias in conversion to one side of the DSB predominated in both mouse and human cells, supporting an SDSA mechanism of HR. Moreover, the donor remained unaltered after HR. Interestingly, while transformed human cells suppressed homeologous recombination, the degree of suppression was less than that observed in mouse cells. For either cell type, BLM deficiency did not alter this suppression, unlike what is observed in yeast Sgs1 mutants. Either other RecQ helicase family members play a role in the suppression of homeologous recombination, or mammalian RecQ helicases do not play a role in this process.