Enzymatic processing of lesions during DNA repair must be coordinated to avoid the secondary creation of intermediates that in themselves would place the genome at risk. While single lesions are efficiently repaired through an SSB intermediate step by the many available redundant BER/SSB repair proteins, closely spaced lesions, if processed at the same time, would be expected to have a high risk of generating DSBs. The presence of nonligatable ends (i.e., “dirty” ends) at SSBs produced during BER might lead to DSBs that cause genome instability and cell death if not efficiently removed (48
). In G1
haploid yeast cells, there is little opportunity for DSB repair through recombination or end joining involving nonligatable ends. In diploid and G2
cells, repair can occur via homologous recombination, although DSB repair capacity is limited in terms of the number of breaks repaired.
While there are extensive studies on the repair of clustered lesions in vitro, using oligonucleotides and purified enzymes or cell extracts, there are relatively few in vivo studies (for a recent review, see reference 22
). Our recently developed in vivo assay has provided a unique opportunity to investigate the formation of DSBs arising from closely spaced lesions during repair of MMS-induced DNA damage in G1
stationary-phase haploid yeast. Since the assay measures clustered lesions in vivo as both SSBs and damaged bases (converted at a high temperature to DSBs in vitro), it is useful for addressing genetic interactions during BER of clustered lesions. The DSBs arising from MMS-induced closely spaced lesions are different from other types of DSBs induced by agents such as ionizing radiation or specific endonucleases, since the BER-associated DSBs are repaired well in G1
haploid yeast even in the absence of Rad52 (42
), which is required for recombinational repair, or Ku70/80 (data not shown), which is required for end joining.
The generation of DSBs by closely spaced SSBs could occur if the overlapping complementary regions are short. Even if lesions are farther apart, repair processes that create SSBs could move SSBs closer to form DSBs, for example, during strand displacement repair synthesis (Fig. ). This was proposed for SSBs produced on opposite DNA strands 80 to 90 bp apart, with an observed repair patch size of 40 nt (56
). If a 5′ flap is not removed by Rad27/Fen1, strand displacement synthesis will continue without ligation, generating even longer flaps capable of binding replication protein A (RPA). Flaps longer than 20 to 30 nt are stably bound by RPA and are prevented from direct cleavage by Rad27/Fen1 (2
); however, such flaps are subject to cutting by Dna2, which usually does not generate ligatable nicks. Strand displacement DNA synthesis would have the effect of bringing more distant ends closer together to generate DSBs. However, long flap formation appears unlikely when Rad27/Fen1 is fully functional during nick translation repair synthesis, since ~90% of the products released by Fen1 are mononucleotides (17
). Thus, the well-coordinated repair of SSBs in WT cells by Pol δ, PCNA, and Rad27/Fen1 would prevent the generation of DSBs via processing of widely (>30 nt) separated lesions.
FIG. 7. Possible mechanisms for DSB formation from closely spaced lesions. When there is coordination of BER enzymes (WT), closely opposed base lesions can be repaired one by one. Once the first lesion is processed by glycosylases followed by AP endonucleases, (more ...)
Although the pol32Δ or rad27-p single mutant had little effect on the repair of MMS damage, not only was the pol32Δ rad27-p double mutant defective in repair of lesions that were closely spaced, resulting in DSBs, but there was also a further increase in these DSBs after LH (Fig. and Table ). Thus, the combination of Pol δ (with the Pol32 subunit) and Rad27 appears to prevent the generation of DSBs at closely spaced lesions. The model in Fig. describes steps in the repair of closely spaced lesions that could lead to DSBs if there is a lack of coordination in BER components, as in the case of the pol32Δ rad27-p mutant. The DSBs could arise either directly after induction of SSBs or during subsequent processing of lesions that are not widely spaced.
Since deletion of MAG1
prevents the accumulation of closely opposed SSBs in the pol32
double mutant (Fig. ), the repair defect leading to DSBs is downstream of an SSB generated by an AP endonuclease. In vitro studies have demonstrated that for pairs of lesions as close as 1 to 3 nt apart, both can be incised by endonucleases/lysases to form a DSB (7
). Whether this can occur in vivo remains to be determined (22
). Our present results showing that the number of closely opposed SSBs was increased during LH in the pol32
mutant (Table ) suggest that simultaneous processing of closely spaced lesions may normally be prohibited in vivo. Possibly, binding of the Polδ-PCNA-Rad27 complex obstructs access of glycosylase and AP endonuclease to nearby methylated bases, resulting in coordinated repair such that the first SSB is repaired before the second SSB is formed. Since Pol32 is required for optimum processivity of Pol δ and may affect strand displacement (10
), the loss of Pol32 might slow the SSB repair process. Therefore, combined with the rad27-p
mutation, the opportunity for generating a second SSB at a nearby lesion may be increased before finishing repair of the first SSB (Fig. ). Also, there would be less of the Polδ-PCNA-Rad27 complex to physically obstruct processing of a nearby second lesion.
The synergy between the rad27-p
mutation, which causes a defect in PCNA binding, and the pol32
Δ mutation reveals the important role of PCNA in the repair of clustered lesions. PCNA interacts with Pol δ and DNA ligase and also forms a tight complex with Rad27/Fen1. This interaction provides efficient removal of 5′ nucleotides by means of “nick translation” to continuously maintain a nick that can be ligated (35
). Displacement of a few nucleotides by Pol δ is enough to lead to removal of the 5′-dRP by Rad27/Fen1 (16
). This may explain why the single pol32
Δ mutation has only a mild effect, since a tight PCNA-Rad27 interaction could efficiently remove 5′-dRPs before a second incision at the nearby lesion or decrease the requirement for strand displacement and/or the length of a displaced flap. In the rad27-p
mutant, a lack of PCNA-Rad27 interaction may lead to deficient recruitment of flap removal activity since the access of Rad27 protein to the site is most likely restricted. However, the reduced access of Rad27 protein to the repair site in the rad27-p
mutant appeared to be compensated for by Pol32, since repair was comparable in the WT and rad27-p
strains (Fig. and Table ). Only when both proteins were defective could the accumulation of DSBs occur.
Pol32 may play a role in helping to recruit the Rad27-p protein to a repair site, possibly by temporally releasing the Pol δ-PCNA complex from the repair site or by helping to generate a suitable flap. Since Pol32 can be involved in TLS (26
), Pol32 may aid in Pol δ strand displacement synthesis past a nearby opposing methylated base, resulting in the generation of a long flap that could be a better substrate for the Rad27-p protein. Pol32 is required in Pol ζ (REV3
)- and Rev1-mediated (but not Pol η-dependent) TLS, at least for the initiation step, to bypass abasic sites (24
). If TLS helps to prevent the accumulation of DSBs, deletion of other TLS proteins in the rad27-p
background should result in MMS responses similar to those for the pol32Δ rad27-p
mutant. However, as shown in Fig. S1 in the supplemental material, this is not the case for the rev1 rad27-p
and rev1 rad27-p
double mutants, suggesting that the synergistic effect between pol32Δ
mutations is not due to a loss of TLS function.
While Pol32 is important for Pol δ processivity and strand displacement, the structural parameters in Pol δ that determine its strand displacement potential remain unknown (16
). However, the Pol32 subunit has an extended structure which accounts for the unusual elongated shape of the entire three-subunit complex (31
). Possibly, this structure helps to “open” the strands to enable bypass of an adjacent lesion without concomitant DNA synthesis. Recent in vitro results show that Pol δ in the absence of the Pol32 subunit has reduced activity for strand displacement (55
Overall, this study and our previous report (42
) reveal that closely spaced single-strand lesions can be converted into DSBs through BER processes in yeast. Closely spaced lesions could result from random lesions that are nearby by chance or from agents such as ionizing radiation, which creates clustered damage. The Pol32 component of Pol δ appears to play an important role in preventing the transition to DSBs by participating in the efficient coordinated repair of closely spaced lesions. It is interesting that Pol32 may also function elsewhere to prevent DSB formation, since the combination of a pol32
Δ mutation with other homologous recombination repair pathway mutations results in slow growth or lethality (25
Clustered lesions may possibly be generated during normal cellular metabolism by reactive oxygen or nitrogen species. The current study with G1 stationary-phase yeast cells has important implications for BER events in G1 human cells, especially postmitotic cells that cannot be replaced through cell division. Prevention of DSB formation by BER might be relevant to issues of neurodegeneration and aging.