These results clearly indicate that following a DSB, a sequence on a heterologous chromosome can serve as a repair template for homologous recombination in mammalian cells, even when homology is constrained. All but one of the clones (79 of 80) we examined had undergone interchromosomal recombination between the neo
sequences without exchange of flanking markers or other genome rearrangement. Instead, repair was initiated by gene conversion and completed by NHEJ. Thus, repair of a single DSB by interchromosomal gene conversion, whether a fully homologous event (40
) or a compound event involving NHEJ as in this report, rarely compromises genomic integrity. The results presented here contrast with the repair of two DSBs in which chromosomal rearrangements (translocations) were readily recovered (39
). In that case, translocations did not arise by gene conversion but rather by joining of the ends of two different chromosomes by NHEJ or single-strand annealing, suggesting that gene conversion has a higher fidelity for maintaining genomic integrity than these other repair pathways.
The importance of NHEJ and homologous repair for the maintenance of genomic integrity in mammalian cells is emphasized by the observations that cell mutants in either repair pathway exhibit a high frequency of chromosomal aberrations (7
). The importance of these two repair pathways is evident in both embryonic and adult cell types, although recent studies suggest that there may be differences between these stages in the contribution of various repair pathways and proteins. For example, ES cells deficient in the homologous repair protein Rad54 are sensitive to ionizing radiation (8
), a potent inducer of DSBs, although this sensitivity seems to decrease through development to the adult mouse (9
). By contrast, adult mice mutant for the NHEJ repair protein DNA-PKcs are hypersensitive to ionizing radiation, although DNA-PKcs−/−
ES cells do not display this phenotype (3
). Nevertheless, mutation of other NHEJ proteins Ku70 and Ku80 leads to ionizing radiation sensitivity in both ES and adult mouse cells (17
), and therefore, it is likely that the Ku protein participates in the NHEJ events that we report here.
NHEJ and homologous repair have also been proposed to have different contributions to repair during different stages of the cell cycle, i.e., G0
. Based on previous work, we expect that the overwhelming majority of repair events at the chr.17 break site are intrachromosomal, involving either NHEJ of the two broken ends or homologous repair from the sister chromatid, which are not selected for in this system (23
). This is supported by the frequency of gene targeting, which is 40- to 240-fold higher than interchromosomal events. What governs the use of a homologous sequence on a heterologous chromosome for repair of a DSB is unclear, but considering the nuclear volume, it is possible that random collision plays a role in homologous partner choice.
The results presented here provide convincing evidence that NHEJ and homologous repair are not completely separable and that coupling of the two pathways can preserve genomic integrity for the repair of a single DSB. Coupling of the two pathways has previously been predicted in some gene targeting events (see, e.g., references 2
, and 41
). This report provides direct evidence for such events and detailed analysis of the junctions. The structure of the recombinant products demonstrates that repair of the DSB was initiated by invasion of one chr.17 end into the homologous sequence on chr.14, priming DNA replication which extended into heterologous sequences. Sequence analysis demonstrated that NHEJ was used to join the newly replicated strands to the other chr.17 end, which in some cases was preserved to such an extent as to maintain the overhang of the break site. The consistent recovery of clones that maintain the other chr.17 end indicates that this end is maintained close to the repair complex even though it does not participate in the homologous invasion step. This coupled repair mechanism can also account for previously observed infrequent LTGC events from allelic and interchromosomal recombination with related substrates (33
), although this has not been verified.
Although similar models for the initiation of recombination have been proposed for yeast (19
) and Drosophila
DSB repair (14
), the coupling of NHEJ and homologous repair appears to occur more readily in mammalian cells and possibly plant cells (38
), presumably due to an overall greater contribution of NHEJ to DSB repair in higher eukaryotes. As a result of this process, the heterologous sequences that are replicated during repair synthesis become duplicated. In most clones we found that the duplication was a few kilobases or less. In none of the clones did replication extend to the end of the chromosome, as has been detected in yeast (30
). However, in mammalian cells replication to the end of the chromosome may lead to inviable progeny, resulting in either unbalanced genetic information (as in the F5′/3′ cell line) or an acentric product (as in the R5′/3′ cell line). Similar constraints on product recovery might also exist if gene conversion with a reciprocal exchange were exclusive to the S/G2
phase of the cell cycle and recombinant chromosomes always segregated from each other.
Although overall genome integrity is maintained in the coupled repair products we observed, the resulting duplication of sequences 3′ to the break site is likely to be deleterious in some cases. Alterations of the ALL1 locus in leukemic cells have been found which involve partial tandem duplications of the ALL1 gene at or near Alu repeats (43
). These duplications mechanistically could have arisen similarly to the events described here (Fig. ). Thus, a DSB within or near a repetitive element could initiate strand invasion into the identical element on the homologue in G0
) or sister chromatid in S/G2
) and prime DNA synthesis. Following repair synthesis, the repair event would resolve by NHEJ (Fig. ). Alternatively, repair synthesis could continue into a downstream repetitive element of the same class, so that the event is resolved by annealing of the newly synthesized strand with the complementary end of the broken chromosome (not shown). The advantage of this model is that it allows for invasion to occur within an identical Alu element or other sequence but has no constraints on the completion of the repair event, since it can occur by either NHEJ or homologous annealing.
FIG. 5 Model for partial tandem gene duplications. Events can be initiated by a DSB within a repetitive element (gray box) on one chromosome, followed by invasion of one end into the same element on the homologue or sister chromatid. Repair synthesis extends (more ...)
It is unclear what minimal length of homology is required to promote interchromosomal homologous recombination in mammalian cells and what effect the length of homology has on crossing over. As little as 68 bp of homology is sufficient for homologous invasion to occur at a detectable frequency during DSB-promoted gene targeting in ES cells, although in this case recombination occurs at a significantly lower frequency than when >200 bp of homology is used (C. Richardson, J. Winderbaum, and M. Jasin, unpublished results). The majority of dispersed repetitive elements, SINEs (<200 bp in the mouse or 300 bp in humans) or small truncated LINEs (as small as 300 bp) (44
), are within the size range of the homologous repeat used in this study. However, LINEs can be longer than this repeat unit. Full-length LI elements are 7 kb, although the majority are truncated to smaller units of a few kilobases or less (44
). It is unclear whether interchromosomal recombination between repeats as long as several kilobases would give rise to repair products different than those reported here.
Surprisingly, we observed a ninefold higher frequency of interchromosomal recombination in the F5′/3′ cell lines in which the neo
sequences are in the same orientation relative to the centromere, compared with the R5′/3′ cell lines in which the neo
sequences are in the opposite orientation, even though the overall structure of the recombinants was very similar for the two cell lines. Cell lines with the other two configurations of the truncated neo
sequences gave similar results (data not shown); for example, a cell line with the neo
repeats in the same relative orientation but opposite to F5′/3′ gave similar recombination frequencies as the F5′/3′ cell line (C. Richardson and M. Jasin, unpublished results). Unless there is loss of a major class of repair product from the R5′/3′ cell lines, these results suggest an unexpected sensing of the relative orientation of the neo
sequences on the two interacting chromosomes during these compound repair events. Rates of interchromosomal Cre/loxP
recombination in yeast have been shown to be affected by centromere clustering (4
), although thus far there has not been a study of this in mammalian cells. It will be interesting to determine if this orientation effect will be generally observed in mammalian cells and, if so, to determine the factors responsible for this phenomenon.