Repair of chromosomal DSBs is essential to maintain genomic integrity, yet different repair pathways are variably mutagenic. In this report, we have examined the interrelationship of the homologous-repair pathways in mammalian cells. HDR is primarily a precise type of repair and thus much less prone than SSA to causing gross chromosomal rearrangements (39
) or chromosomal deletions, as assayed in this study. However, HDR can lead to genetic loss under some circumstances, such as when conversion is extensive. Thus, the proper balance and regulation of DSB repair pathways would be predicted to be critical for the maintenance of genomic integrity. In support of this, HDR mutants exhibit chromosome aberrations and higher rates of mutagenesis (48
), apparently as a result of misrepair of spontaneously arising DNA lesions, including DSBs. As yet, however, a comparative analysis of distinct classes of DSB repair mutants has not been reported to provide an understanding of the interrelationship of the homologous-repair pathways in mammalian cells.
Our analysis has determined that some factors affect the competitive choice between HDR and SSA (RAD51 and BRCA2), whereas others factors promote or suppress both homologous-repair pathways (BRCA1/BARD1 and KU, respectively), and we have identified an activity that limits genetic loss from HDR (ATP hydrolysis by RAD51) (see below) (Fig. ). Failure to specifically utilize HDR as a result of impaired RAD51 function—either directly by RAD51 mutation or indirectly by BRCA2 disruption—leads to an increased reliance on SSA. The RAD51 ATP hydrolysis mutant not only shifts homologous pathway use to SSA but also fails to limit the extent of gene conversion in the remaining HDR events, increasing the potential for genetic loss.
FIG. 7. Genetic steps of homologous repair with distinct mutagenic consequences. Shown are different pathways of DSB repair and the roles of individual factors in pathway choice. Whereas homologous repair by HDR with limited gene conversion (blue) is a precise (more ...)
The finding that the proper functioning of RAD51 is essential to promote precise homologous repair indicates that control of strand exchange may be a deciding factor in suppressing mutagenic outcomes of DSB repair. BRCA2 is a particularly intriguing factor for such control, since it can interact directly with RAD51 at multiple motifs, as well as with single-stranded DNA, and thus may modulate the association of RAD51 with damaged DNA (7
). Interestingly, we found a shift from HDR to SSA, whether BRCA2 is mutated by the loss of only the C-terminal RAD51 binding motif so that 94% of the protein coding region is left intact, including the eight BRC repeats (23
), or whether a single 70-amino-acid BRC repeat, which can disrupt RAD51 nucleoprotein filament formation in vitro, is overexpressed. Another Brca2
mutant, as yet uncharacterized, also shows increased deletional homologous-recombination events which may reflect SSA (23
). Thus, a shift from HDR to SSA appears to be a generalized outcome when RAD51 or BRCA2 function is disrupted. Consistent with this, loss of RAD54, a protein that promotes RAD51-mediated strand exchange activity in vitro (51
), also results in a shift from HDR to SSA (11
), although the magnitude of the shift is much smaller than that seen with RAD51 or BRCA2. Thus, this shift in homologous pathway usage appears to be a common outcome when factors that affect steps after the branch point of HDR and SSA are disrupted. Although key distinctions exist for some yeast and mammalian DSB repair factors, a similar shift to SSA has been noted in yeast for some HDR mutants (16
). The shift to SSA may result from hyperresection of DSBs when RAD51 activity is impaired: whereas HDR requires minimal resection, SSA requires >2 kb of resection in our substrate. Since repetitive elements are at various distances from each other, the extent of resection is expected to be an important factor in the efficiency of SSA in the mammalian genome.
In addition to the shift to SSA, we found that genetic loss arising from more extensive gene conversion tracts also occurred when ATP hydrolysis by RAD51 was disrupted. Mutation of the RAD51 paralog XRCC3 also results in more extensive gene conversion in the residual recombinants that are obtained with this mutant, although the tracts are often discontinuous (5
). As we find no evidence for discontinuous tracts with RAD51-K133R expression, RAD51 and XRCC3 appear to have different roles in the extent of conversion. Perhaps XRCC3 is essential to promote the stability of recombination intermediates and thus maintain the continuity of DNA synthesis during gene conversion, whereas ATP hydrolysis by RAD51 is important for processing recombination intermediates to limit such DNA synthesis. Alternatively, ATP hydrolysis by RAD51 may result in greater protection of DNA ends, limiting the amount of single- or double-strand resection that may occur.
In contrast to RAD51 and BRCA2, we found that BRCA1/BARD1 or KU70/KU80 affected both homologous-repair pathways in the same direction; defects in BRCA1/BARD1 decreased the efficiencies of both homologous-repair pathways, while loss of KU increased the efficiencies of both pathways. Thus, these factors appear to affect a step(s) of homologous repair that is common to both HDR and SSA, although with opposite outcomes. Furthermore, loss of KU70 partially suppresses the homologous-repair defects of BARD1 disruption, which indicates that BRCA1/BARD1 and KU70 may both be acting at an early step during homologous repair. The initial strand resection step to generate single-stranded DNA for strand exchange or annealing is one such step. Ku70-deficient yeast cells exhibit elevated rates of strand resection, indicating that Ku70 is important in limiting resection in yeast (24
). Thus, while KU70 could limit strand resection, BRCA1/BARD1 could act to promote strand resection. An early role for the BRCA1/BARD1 complex in homologous repair is consistent with its rapid localization to DSBs (35
). The exonuclease(s) that promotes resection has not been identified; however, BRCA1/BARD1 could affect nuclease activity or access of a nuclease to DNA ends via one of the identified BRCA1/BARD1 biochemical activities, e.g., DNA binding, ubiquitination, or phosphopeptide binding (6
). Unlike SSA, the inhibitory effect of BARD1 disruption on HDR is not totally abrogated by Ku
mutation; thus, BRCA1/BARD1 could still have a later role in HDR, possibly through its interaction with HDR-specific factors like RAD51 or BRCA2 (18
We also determined that mammalian RAD52 promotes SSA, although loss of RAD52 does not overtly affect HDR. That RAD52 does not play a significant role in promoting HDR in mammalian cells is surprising, given its critical role in HDR in yeast (52
), its postulated role as a “gatekeeper” for HDR in mammalian cells through its biochemical activity of binding to DNA ends (56
), and the fact that human RAD52 stimulates homologous pairing by human RAD51 in vitro (51
). The nonessentiality of RAD52 in mammalian cells suggests this function is assumed in vivo by some other factor. BRCA2, which also binds single-stranded DNA and RAD51 and stimulates RAD51 activity (7
), is an excellent candidate. However, a role for RAD52 in HDR may still be uncovered in other, more complex genetic contexts, as has been observed in chicken cells by combined mutation with the RAD51 paralog XRCC3 (13
In summary, the multiple DSB repair pathways have a complex interrelationship which affects whether repair occurs faithfully. Understanding these interrelationships at the molecular level in mammalian cells is essential, given that many DSB repair mutants have markedly different phenotypes, including development defects, aging phenotypes, and tumor susceptibility (38
), and in some cases even have tumors of the same tissue type yet with markedly different characteristics, i.e., as in humans for BRCA1- and BRCA2-associated tumors (14