We have summarized the data obtained in the Results section in and the strains have been placed into three groups on the basis of sensitivity to methylating agents. A striking result is the sensitivity of strains with single mutations in genes affecting recombination (recA, priA) compared with those affecting BER (ada, tag, ogt). Apart from the BER-defective alkA tag double mutant, this correlation becomes more striking for cells with multiple mutations affecting recombination (e.g. recBCD recF, recBCD alkA tag). We conclude that homologous recombination is essential for the repair or tolerance of methylated lesions in DNA.
Sensitivity of GM7330 derivatives to acute MNNG and MMS exposure
In addition to conditions where cells were exposed to an acute low dose of methylating agent, we have also tested the effect of chronic exposure by growing cells on solid media for 2 days in a concentration gradient of MMS or MNNG (‘gradient plates’). The results were in agreement with those for acute exposure (–) except for the strains with ada-alkB, alkA, ada ogt and xthA nfo mutations which showed a greater sensitivity after chronic versus acute exposure (data not shown). The recF strain, on the other hand, was more resistant to chronic than acute exposure to methylating agents. We also found that overexpression of Tag or Ada did not change survival in recBCD cells (data not shown).
It is remarkable that strains deficient in recombination, such as recA
, recBCD recF
and ruvC recG
, are as sensitive to methylating agents as BER repair-deficient alkA tag
bacteria. This observation indicates that recombination is as important as BER for cells to repair damage inflicted by MMS and MNNG. Both RecBCD and RecFOR pathways are required () but since the survival of the recBCD recF
double mutant is less than each alone, they act independently of each other. This is not unexpected given that RecBCD is involved primarily in DSB repair and RecFOR in the repair of gaps (15
). The requirement for both of these recombination systems is compatible with at least two types of methylation-induced damage requiring recombinational repair. Although DNA single-strand breaks have been detected after alkylation damage, there is no evidence that these agents directly form DSBs. It is more probable that the single-strand breaks or replication-blocking lesions are converted to DSBs during DNA replication.
DNA single-strand breaks could arise in methylated DNA by at least two mechanisms. First, AlkA or Tag glycosylase action forms abasic sites and then AP endonuclease activity cleaves the DNA backbone. Second, abasic sites that are produced after spontaneous 7-meG depurination eventually become substrates for AP endonucleases. The independence of AlkA or Tag-mediated BER from homologous recombination suggests that the second possibility is more likely. It is probable that nicks generated by AP endonucleases, which require additional processing by deoxyribophosphodiesterases, are longer lived than simple ligatable nicks with a 3′-OH and a 5′-phosphate and so there is a greater chance that they will be encountered by a replication fork. If these nicks are encountered by a replication fork, they will produce replication fork collapse, generating substrates for RecBCD pathway recombination (43
). During re-construction of the fork, the PriA pathway proteins reload the DNA polymerase III holoenzyme (37
). This explanation could account for the sensitivity of priA
mutants to methylation-induced toxicity, since such cells would be less capable in restoring collapsed replication forks ().
Although it is clearly the case that a single-strand break formed during BER could potentially cause replication fork breakdown, unrepaired base damage is also potentially recombinogenic. For example, unrepaired 3-meA can block replicative polymerase progression (5
). Indeed, we observed that there is an enormous increase in the sensitivity of recBCD
mutant cells when AlkA and Tag are deleted, presumably because unrepaired 3-meA causes formation of DSBs. Similarly, abasic sites arising from N7-methylguanine depurination, for example, also block replicative polymerase progression (44
). The viability of xthA nfo recF
cells and their poor survival after MMS exposure () is consistent with RecBCD-promoted DSB repair at stalled replication forks at abasic sites. The inviability of xthA nfo recBCD
bacteria () is probably due to replication fork problems at spontaneously occurring abasic sites or blocking lesions. We speculate that this effect is magnified after methylation damage based on the increased susceptibility of xthA nfo
cells chronically exposed to MMS and MNNG. Regardless of the underlying mechanism of methylation-induced recombination, the data presented here are consistent with both unrepaired methylation damage and BER repair intermediates inducing DSBs.
Inviability of recB AP endonuclease-deficient bacteria
Our conclusions regarding the role of AP-endonucleases in promoting recombinational repair are similar to those reached by Wang and Chang (23
). These authors found that xthA nfo nth
(endonuclease III) mutants were more sensitive than wild-type to MMS and that recA
, derivatives could not be constructed but that a recF
derivative could be. Although there are some differences between their data and ours, we agree that methylated bases produce secondary lesions that require the function of the recA
genes to ensure survival.
Our observations suggest that there is a difference between MNNG and MMS in recombinational repair of DSBs and gaps. The most compelling data are the responses of the alkB recF
() and xthA nfo recF
cells (). The alkB recF
cells show wild-type resistance to MNNG but high sensitivity to MMS. The xthA nfo recF
bacteria are more sensitive to MMS than MNNG suggesting that DSB repair is more important after MNNG damage than gap repair and vice versa for MMS. The basis for these differences between MNNG and MMS is not clear as the amounts of N-methylated residues formed in DNA are not very different (12
) except for 4-fold increases in N1-methyladenine and N3-methylcytosine (AlkB substrates) by MMS versus MNNG. It is clear from these results, however, that the requirement for RecF recombination is greater in cells exposed to MMS than MNNG. MNNG produces more O-methylated bases than MMS which probably explains why ada ogt
cells are more sensitive to MNNG than MMS () and may indicate that these O-methylated bases act as lethal lesions as previously suggested (29
). This suggestion is supported by our data in and which, because of the wild-type survival of ada
-regulated gene disruptions (alkA
), can be interpreted as indicating that it is the methyltransferase, and not the regulatory function, of Ada that is important. We speculate that, at least some of the time, O-methylated bases block progression of the replication fork and promoting fork stalling, collapse or breakage. The results with the ada ogt
and alkA tag
double mutants suggest that AlkA, Tag, Ada and Ogt are all required to promote survival after MNNG challenge while survival after MMS exposure is dependent predominantly on the alkA
genes products (). These results are consistent with previous observations (1
One unexpected and very interesting result of these studies was the observation that polA
mutant cells are extremely sensitive to methylating agents (). Although MMS sensitivity of polA
mis-sense and nonsense mutants has been described previously [e.g. (41
)], the degree of killing was much less than that for the polA
deletion mutant. This result suggests that PolI may have functions other than simply nick translation in BER or gap filling in NER. PolI is essential for intrachromosomal recombination initiated by a DSB (39
) and, therefore, it could also be required for DSB repair after methylation damage to DNA. Our data () suggest that both 3′-5′ exonuclease and Klenow fragment functions are required for resistance to methylation damage.
The increased sensitivity of the alkA tag
double mutant versus the single mutants likely reflects the redundancy of the glycosylases in repairing damage. Alternatively, the decreased survival might indicate that when replication-blocking methylated bases persist in DNA, recombination becomes a major mechanism for the cell to tolerate such lesions. This explanation is similar to that proposed for the repair of ultraviolet damage in uvr
mutants deficient in NER (48
) where single-strand gaps occur on both leading and lagging strand which have to be filled by recombination. The data presented here suggest that such gaps are also formed after methylation damage and repaired by the RecFOR system. These gaps could arise at a blocking lesion by decoupling leading and lagging strand polymerase assemblies (46
) allowing further lagging strand synthesis followed by re-initiation of replication after PriC loading of DnaB (50
We were surprised to observe that the uvrD
strain showed moderate sensitivity to MNNG (). The UvrD helicase performs essential steps in NER and mismatch repair (MMR). Since NER is not involved in repair of methylated lesions (consistent with the fact that disruption of the uvrA
gene does not sensitize cells to methylation damage, ) and MMR effects occur predominantly in dam
mutants, it is unlikely that the sensitivity of the uvrD
mutant is because of these processes. Alternatively, it is possible that UvrD helicase action may be necessary for DSB repair by recombination as UvrD-deficiency has been reported to affect recombination (51
). In addition to its involvement in NER and MMR, UvrD has recently been implicated in replication fork reversal (52
). These results, therefore, suggest the possibility that fork reversal at replication-blocking lesions, such as 3-meA, may require UvrD or that this helicase is required for recombination during repair of methylation-induced DSBs.
In conclusion, our results suggest that recombination is essential to repair both DSBs and single-strand gaps after methylation damage. DSBs result from replication fork problems at single-strand nicks resulting from AP-endonuclease action, and from replication-blocking lesions such as 3-methyladenine, abasic sites and O-methylated bases. A prediction of the genetic data presented in this paper is that DSBs should be formed in cells exposed to MNNG as a consequence of DNA replication and we are currently seeking evidence for this prediction.