We have examined the effect of heterology on DSB-induced homologous recombination in mammalian cells, comparing wild-type and Msh2−/−
ES cells. We found that in wild-type cells, the frequency of recombination between diverged sequences in a gene-targeting assay was reduced as the sequence divergence increased, consistent with what was previously observed (17
), but that the effect of heterology was significantly overcome in Msh2−/−
cells. Thus, for a plasmid substrate with 1.5% heterology relative to the chromosomal sequence, DSB-induced recombination is reduced 17-fold in wild-type cells when normalized to recombination with a nearly identical substrate, but only 1.7-fold in Msh2−/−
cells. Similar to gene targeting, DSB-induced intrachromosomal recombination between diverged sequences was also reduced to a greater extent in wild-type cells than in Msh2−/−
cells. The products of recombination in the Msh2
mutant were also significantly altered. Most notably, the majority of GCTs derived from intrachromosomal recombination had mixed polymorphic markers present in the homologous repair substrate, implying that hDNA formation followed by MMR correction is a major mechanism for the conversion of markers near a DSB in mammalian cells.
These results are in agreement with those previously reported for mammalian cells in which the barrier to spontaneous recombination between diverged sequences was found to be relaxed in MMR-deficient cells (1
). The fold reduction of recombination in wild-type cells in these experiments was similar to or even greater than what we saw in the wild-type cells with our most diverged substrate. However, in our experiments we found that recombination was not fully restored in the Msh2−/−
ES cells, whereas in the previous reports the barrier to recombination was found to be almost completely abrogated by Msh2
mutation. This difference may be attributable to the design of the gene-targeting experiments. Our experiments examined DSB-induced recombination of small DNA fragments containing single nucleotide substitutions, whereas the previous experiments investigated recombination in the absence of induced chromosomal damage using much larger fragments that contained different types of polymorphisms.
In addition to the frequency of recombination, we have also examined GCTs in the recombinants derived from the Msh2 mutant cell line. This is the first time, to our knowledge, that GCTs in mammalian cells in an MMR-defective background have been studied. For this study, we examined phenotypically silent mutations spaced every 50 to 100 bp or less in the repair substrate. DSB repair by gene conversion has been proposed to occur by two possible mechanisms: (i) hDNA formation followed by mismatch correction and (ii) gap formation after both strands adjacent to the DSB are processed. Examination of the GCTs in cells which lacked MMR activity allowed us to infer which mechanism of gene conversion was occurring during DSB repair in wild-type cells. We observed that 55% of Msh2−/− neo+ recombinants had mixed GCTs, which were predicted by hDNA correction, whereas only 2.5% of the wild-type recombinants had mixed GCTs. Considering the silent mutations individually, most mutations when converted were partially converted in the recombinants (i.e., mixed; Fig. ), suggesting that the conversion involved hDNA correction. The mutation that was the one exception was the one nearest the DSB (the NsiI site), which is frequently fully converted, even in Msh2−/− cells. Thus, with the exception of the mutation at the NsiI site, 39 of 48 (81%) conversion events involved hDNA formation. The frequency for each mutation depends on the distance from the DSB site, since mutations at some distance from the site (≥183 bp) were found to be incorporated exclusively by hDNA correction (Fig. ).
Summary of gene conversion frequencies for each silent mutation in wild-type and Msh2−/− cells. The conversion frequency for each mutation (Fig. ) was plotted as a function of distance from the DSB.
Gap formation was expected to account for the remaining conversion events. Fully converted mutations were anticipated to occur in the Msh2
mutant only if both strands of the DNA adjacent to the DSB were degraded by nucleases. Information for the repair would then have come solely from the diverged donor substrate. In three conversion events, gap formation may have extended 100 bp or more from the DSB. Gap formation, however, was especially evident at the Nsi
I site located 8 bp from the I-Sce
I site, since it was fully converted in 70% (58 out of 83; Fig. ) of the conversion events (Fig. ). In the remaining events at this site, processing at the DNA ends led to the loss of the I-Sce
I site but also to the retention of nucleotides only 8 bp further away, although it is not clear if the sequence heterology of the I-Sce
I site affected the incorporation of this mutation. In summary, therefore, we infer that gene conversion occurs primarily by hDNA correction, but that sequences near the DSB can be incorporated by gap formation, the frequency depending on the distance from the DSB. These results are generally in good agreement with those obtained with yeast, in which it has been found that sequences close to the end of a DSB are preserved during gene conversion (22
), although the results here suggest that there may be somewhat more nibbling of ends in mammalian cells. Evidence for hDNA correction during DSB-promoted and spontaneous gene conversion in wild-type mammalian cells has previously been obtained using palindromic markers which are not efficiently repaired by MMR (15
). However, some other aspects of the GCTs (i.e., length and discontinuity) differ from results obtained here with single polymorphisms.
Current models for mitotic gene conversion favor mechanisms in which recombination is coupled to replication (3
). In these models, a 3′ end invades the unbroken homologous template and initiates repair synthesis. The newly synthesized strand is then incorporated into the broken molecule, which can result in hDNA formation. In principle, invasion of either one or both 3′ ends flanking the DSB could initiate repair synthesis. Our bidirectional GCTs are consistent with invasion of the template from both 3′ ends, since markers on each side of the DSB segregate at the next cell division (Fig. ), as would be expected from hDNA formation involving opposite strands. The majority of recombinants (27 out of 40; 68%) did not show bidirectional tracts, however. In these cases, two-ended invasion may have occurred but may not have incorporated the polymorphic markers. Alternatively, one-ended invasion may have occurred, as has been proposed for other mammalian gene conversion events (24
Our results also indicate a trend toward longer GCTs in the Msh2
mutant. In 80% of the wild-type recombinants, the observed GCTs were 58 bp or less, consistent with previous results (17
), whereas in the Msh2−/−
recombinants, only 52% of GCTs were as short. We saw a sixfold increase in the Msh2−/−
cells in GCTs extending to the most distant 3′ polymorphism from the break site, Pml
I. We also saw for the first time the incorporation of the most distant 5′ polymorphism, Apa
I, in one of the mutant recombinants. The mean observed GCT was also found to be somewhat longer (by 20%) in Msh2−/−
cells than in wild-type cells. Two mechanisms could account for the longer GCTs: (i) MMR proteins may regulate the length of hDNA formation, as suggested previously (7
), or (ii) the length of hDNA is the same, but in wild-type cells, correction of hDNA at a distance from the DSB is in favor of the recipient DNA molecule rather than the donor. Support for the latter mechanism is that the strand copied from the donor molecule would be expected to be broken at the end of the hDNA tract, and strand breaks are known to influence the direction of mismatch correction. Nevertheless, in bacteria, MMR proteins have been shown to inhibit RecA-catalyzed strand transfer (59
), consistent with a direct effect on hDNA length.
In MMR-deficient bacteria, gross chromosomal rearrangements involving diverged DNA are increased as a result of the recombinator phenotype of these mutants (43
). Results in this report, as well as those previously published, demonstrate that MMR-deficient mammalian cells also have increased recombination as a result of a relaxation of recombination between diverged sequences. These results would seem to predict that MMR-deficient mammalian cells, like bacteria, would exhibit frequent gross chromosomal rearrangements. Surprisingly, tumors derived from HNPCC patients are generally euploid and do not display gross chromosomal structure defects, even while cell lines from sporadic tumors show continuous chromosomal instability (reference 4
and references therein; 18
). Possibly, more subtle chromosomal rearrangements than those that would be apparent from chromosome number analyses are present in MMR-deficient cells but have yet to be detected.
Nevertheless, several factors may contribute to the maintenance of a normal genotype in cells with MMR deficiency. First, the barrier to recombination between diverged elements is not completely overcome. Although DSBs induce the highest levels of recombination detected thus far (32
), DSB-induced recombination between sequences that are only 1.5% diverged are still not fully restored, even with MMR mutation. Considering that repetitive elements in mammalian genomes are often significantly more diverged (e.g., an average of 15% for Alu
), recombination between highly diverged elements is predicted to be suppressed at least partially in MMR-deficient cells. Second, the outcome of DSB-induced recombination in mammalian cells has a strong bias towards noncrossover events (24
). Therefore, recombination between repetitive elements would not be predicted to have a discernible outcome in most instances because there would not be an exchange of flanking markers. Third, in addition to having a role in recombination between diverged sequences, MMR proteins in yeast are known to have a second role in recombination, such that their mutation would decrease recombination instead of leading to its enhancement. This role involves the removal of nonhomologous tails which are 30 nucleotides or longer and which are formed during some types of recombination (41
). Because dispersed repetitive elements are flanked by DNA which is not homologous, initiation of recombination outside two dispersed elements, unlike the events assayed in this report, is likely to lead to the formation of nonhomologous tails involving the flanking DNA. Although this role for MMR proteins has yet to be demonstrated in mammalian cells, efficient removal of these tails in yeast requires Msh2 (41
), suggesting that recombination between dispersed elements could be reduced by Msh2
Despite these multiple levels of control that maintain chromosome integrity in MMR-deficient cells, the experiments presented here nevertheless demonstrate that these cells have a recombinator phenotype for DSB-induced events, raising the possibility that these cells undergo promiscuous recombination at some level. Although tumorigenesis in HNPCC has been clearly linked to the mutator phenotype arising from an inability to repair replication errors (20
), recombination between diverged sequences in MMR-deficient cells may contribute to the genomic alterations important for the development of the disease. Additional experiments will be necessary to further delineate the effect of MMR mutation on homologous recombination in mammalian cells.