In this study we have thoroughly characterized rad52
class C mutants, which were originally identified in a plasmid-based screen as being γ-ray sensitive but proficient for HR [17
]. First, we confirmed this separation-of-function phenotype in strains where the mutations were integrated into the RAD52
locus. Next, we examined the separation-of-function phenotype in more detail. With respect to HR, we showed that the mitotic HR observed in rad52
class C mutants is not due to a compensatory effect of Rad59. With respect to the inability of rad52
class C mutants to repair DNA DSBs, we analyzed them in three different types of DSB repair assays, one measuring repair by SSA (direct-repeat recombination assay) and two measuring repair by gene conversion (mating-type switching and plasmid gap-repair assays) and firmly established that these mutants are defective in DSB repair by HR. Indeed, the repair efficiencies of the three different types of DSBs in the rad52
class C mutants resemble that obtained in the absence of Rad52. Moreover, the results obtained with the plasmid gap-repair assay, where the individual contributions of HR and NHEJ to DSB repair can be evaluated, show that a gapped plasmid is repaired preferentially by NHEJ in rad52
class C mutant strains rather than by HR as in wild-type strains. Thus, we conclude that rad52
class C mutants fail to repair endonuclease-induced DSBs via mechanisms that require strand invasion of an intact homologous sequence as well as via a more simple mechanism where the ends can be joined by annealing.
Several of the Rad52 functions in DNA DSB repair could potentially be impaired by the rad52
class C mutations as Rad52-mediated DNA DSB repair is a multi-step reaction that involves many activities, including binding to Rad51, Rad59, RP-A, and DNA [46
]. All nine rad52
class C mutations are situated in the evolutionarily conserved N-terminus of Rad52, which contains a DNA-binding domain, domains responsible for Rad52 self-association, and Rad59 binding [50
]. However, an inspection of the three-dimensional crystal structure of an N-terminal fragment of human Rad52 [52
] showed that eight of the corresponding amino acid residues in the human Rad52 structure are located in, or close to, the putative DNA-binding groove (see Figure S2
). The remaining amino acid residue, HsRad52-Y81 (ScRad52-Y96A) is buried beneath this groove. Moreover, four of the corresponding human Rad52 mutant species: HsRad52-Y51A (ScY66A), HsRad52-R55A (ScR70A), HsRad52-R70A (ScR85A), and HsRad52-Y81A (ScY96A) have been purified, and the three latter species show decreased affinity for single-stranded DNA [52
]. Hence, the failure of Rad52 class C mutants to perform efficient DSB repair may likely be a consequence of impaired DNA-binding activity.
Defective DNA-binding of Rad52 may affect several stages of the DNA repair process, e.g., resection, homology search, strand invasion, and second-strand capture. The genomic blot analyses indicate that the nucleolytic processing of DSB ends is intact in rad52
class C mutant strains. This is not surprising, as most models for DSB repair predict that the single-stranded DNA tails at the break are covered by RP-A before Rad52 is recruited [56
]. In agreement with this view, we have previously shown that RP-A is recruited to a DSB in the absence of Rad52 [60
]. In fact, if Rad52 class C mutants are defective in DNA binding, the inability to repair DNA DSBs could simply be explained by the failure of the mutant proteins to recognize and bind to these lesions. In contradiction to this view, we observed in two independent experiments that Rad52-C180A was recruited to defined DNA DSBs, one induced by the HO-endonuclease on Chromosome III and one induced by I-Sce
I on Chromosome V. These results indicate that the DNA-binding domain in the Rad52 N-terminus is not required for Rad52 recruitment to the DNA lesion. This recruitment is then most easily explained by a scenario where Rad52 is attracted to the DNA lesion via its ability to interact with RP-A when the latter has formed a complex with single-stranded DNA at the DSB. This view is supported by the observation that Rad52-C180A, like wild-type Rad52, physically interacts with Rfa1 in a two-hybrid assay (unpublished data) and that no wild-type Rad52 focus is formed at a lesion in the absence of RP-A [60
]. Accordingly, rad52
class C mutations affect a function in Rad52 that is downstream of damage recognition. In fact, we have shown that the reaction is blocked after Rad51 has been recruited to the DNA DSB. However, our experiments do not show whether Rad51 or Rad52 are bound to the damaged DNA in the repair center. The possibility exists that they are just attracted to the lesion via protein-protein interactions. In this case, the failure of Rad52 class C mutants to efficiently bind DNA could result in its inability to mediate the replacement of RP-A by Rad51, thus impairing repair. If, on the other hand, a Rad51 filament is formed at the lesion in rad52
class C mutant cells, we speculate that the DSB remains unrepaired either because the defective Rad52 DNA-binding activity impairs a Rad52-catalyzed homology search important for strand invasion or an annealing step important for second-strand capture. The latter view may explain the observation that the BIR efficiency is reduced only 3-fold in rad52-C180A
strains as second-strand capture is not required in BIR.
It is generally believed that DSBs are the lesions that initiate spontaneous HR. However, here we show that nine rad52
class C mutants are proficient for spontaneous inter- and intrachromosomal heteroallelic HR, but fail to repair different types of DSBs. Since Rad52 forms spontaneous repair foci during S-, but not during the G1-phase of the cell cycle [38
], an alternative source of spontaneous HR could be recombinogenic DNA ends generated when a migrating replication fork converts a nick into a DSB. Such ends may be easier to repair than those induced by γ-irradiation and endonucleases as they require only a one-ended invasion of the intact strand to restore the replication fork. However, rad52
class C mutant strains are sensitive to camptothecin and Top1-T722A expression. If the replication-induced ends in these experiments are equivalent to the DNA end rescued in the BIR experiment, these results may appear surprising as BIR is reduced only 3-fold in rad52
-C180A compared to wild-type. However, unlike in the BIR experiment, multiple lesions are likely produced when Top1-T722A is expressed and in the presence of camptothecin, and strains with a weakened, but not abolished, ability to repair such lesions may die. The possibility therefore still remains that one-ended DNA breaks contribute to spontaneous HR. However, if this contribution was large, one would expect that even a small, but significant, reduction of the efficiency of one-ended DNA break repair should be reflected as reduced HR levels. Since this is not the case in the rad52
class C mutants, replication-induced breaks are likely to initiate only a minor fraction of the spontaneous recombination events.
Based on the above, we find it unlikely that DSBs contribute substantially to the spontaneous HR observed for rad52
class C mutant strains, since mutant cells only rarely survive even a single DSB. This view is supported by work from Fabre and colleagues based on their studies of srs2
]. They argued that srs2 sgs1
synthetic lethality is due to a toxic recombination intermediate. If this intermediate were initiated by spontaneous DSBs, they must occur at a sufficiently high frequency to prevent propagation of srs2 sgs1
strains. However, since rad52 null lig4
as well as rad52 null lig4 srs2 sgs1
strains, which cannot repair DSBs (i.e., no HR and no NHEJ), are viable, they conclude that DSBs cannot be initiating the frequent recombination intermediates that kill srs2 sgs1
strains. In this context, it is important to note that around 75% of rad52-C180A
cells spontaneously develop a Rad52-C180A focus during the cell cycle. If these foci solely represent attempts to repair DSBs in the genome, then rad52-C180A
strains should be inviable due to their inability to repair DSBs. Moreover, we observe that Rad52-C180A foci are turned over before cell division, albeit at a slow rate, suggesting that repair of the spontaneous lesions is in fact completed.
We also note that some of the rad52
class C mutants are hyperrecombinogenic. This behavior is similar to mutations in proteins of the Mre11-Rad50-Xrs2 complex (MRX), which act at an early stage of both HR and NHEJ [3
], and also produce a hyper-recombination phenotype [62
]. The high HR rate in these MRX mutants is thought to be due to a shift in the preferred repair template from the sister chromatid to the homologous chromosome, hence increasing the number of scorable recombinants [63
]. We find that the median lifetime of a Rad52 class C mutant repair focus is seven times longer than a wild-type Rad52 focus. This longer time frame of repair may increase the frequency of genetic exchange with the homolog. Alternatively, more recombinational lesions may be formed in rad52
class C mutant strains. In fact, we observe that a larger number of cells spontaneously form Rad52 repair foci during the cell cycle in rad52
class C mutant cells than in wild-type cells.
If only a minor fraction of spontaneous HR is initiated by DSBs, alternative lesions need to be considered as triggers for HR. In many of the original models for HR, DNA nicks and single-stranded gaps were proposed to initiate HR [64
]. In the present study, we find that UV-irradiation leads to a dramatic increase in interchromosomal heteroallelic HR in rad52
class C mutant strains. This is similar to what has been observed for wild-type strains. UV-rays mostly produce pyrimidine dimmers, and in the dark, the majority of these lesions are repaired by the nucleotide excision and base excision repair pathways. This type of repair produces nicks and single-stranded DNA gaps that could be recombinogenic. Moreover un-repaired pyrimidine dimers may lead to stalled replication forks and expose regions of single-stranded DNA. Single-stranded gaps are potent substrates for recombination in E. coli
via the RecFOR pathway [13
], and it is interesting to note that both the RecFOR complex and Rad52 mediate replacement of a single-strand binding protein, SSB and RP-A, respectively, to allow access of a protein with a strand invasion activity, RecA and Rad51, respectively, during DNA repair [16
]. In addition, a stalled replication fork may produce a DNA substrate suitable for HR, if the fork is regressed into a “chicken foot” structure and the resulting DNA end processed by nucleases to produce a stretch of single-stranded DNA [30
]. Considering that Rad52 repair foci form during DNA replication, such lesions are attractive candidates as substrates that elicit spontaneous HR.
Recently, it was demonstrated that nicked intermediates produced by mutant RAG proteins during V(D)J recombination can be channeled into HR [70
]. Furthermore, the spectrum of spontaneous recombinants obtained in an assay that measures direct-repeat gene conversion and unequal sister-chromatid exchange in a mammalian cell line is similar to the spectrum obtained after addition of camptothecin, but different from the spectrum obtained after induction of recombination by the endonuclease I-Sce
]. Accordingly, lesions other than DSBs may also play a significant role in spontaneous HR in higher eukaryotes.