MMR-induced DSBs in cisplatin-treated cells arise by a replication-dependent mechanism (), most likely involving replication fork collapse () and there is a direct correlation between the presence of these DSBs and cytotoxicity [14
]. MMR-mediated sensitization of dam
cells can also be formed by base analogs such as 2-aminopurine [10
] which requires DNA replication for its cytotoxic action. It seems reasonable, therefore, that MMR-mediated DSB formation in MNNG exposed dam
cells should also be replication-dependent. In contrast, we found no dependence on chromosome replication using cells that are actively synthesizing DNA and those which are not (). Even with lower or higher MNNG doses, we could not detect a replication-dependent effect (data not shown).
To explain this unexpected result (), we suggest that there are at least two different mechanisms for the formation and repair of MMR-induced DSBs. One of these requires chromosome replication just like base analogs or cisplatin and probably involves replication fork collapse but the number of such DSBs is masked by those arising independent of DNA replication. Our inability to detect these replication-dependent DSBs may be due to the limitations of the PFGE assay since we have shown recently that single cell microgel electrophoresis can detect DSBs that are below the limit of detection with PFGE [38
]. In addition, it may be that MMR gaps induced at cisplatin lesions are more stable than those induced by MNNG thereby increasing the probability of an encounter with a replication fork.
Fig. 10 Proposed model for DSB formation in MNNG-treated dam cells. (A) MNNG damage to DNA produces heat-labile (HL) sites that are rapidly and efficiently repaired by (short-patch) BER in the absence of MMR. (B) In the presence of O6meG, MMR can produce excision (more ...)
Replication-independent DSBs might arise as a consequence of interference between MMR and base excision repair (BER) at abasic sites which can be formed in GM56 Δada
by the spontaneous depurination of 3-methyladenine and 7-methylguanine and basal Tag glycosylase activity acting principally on 3-methyladenine residues. We have shown previously [25
] that the contribution of AlkA glycosylase is minimal at (a) the low dose of MNNG (10 μg/ml) and short time of exposure (10 min) and (b) in the absence of Ada, a positive regulator for alkA
gene expression [30
]. These abasic sites are repaired by BER, which results in short repair patches of only a few nucleotides, on both DNA strands thereby ensuring the integrity of duplex DNA (). In MMR-proficient GM56 Δada
, however, some O6
-meG/C base pairs are bound by MutS (such binding was demonstrated in the antirecombination assay in vitro [39
]) and MMR gaps are formed which can be hundred or thousands of nucleotides in length. During this process we speculate that a BER single-strand nick or gap is encountered on the complementary strand leading to the formation of a DSB (). DNA replication would not be required for DSB formation () and the DSBs formed this way could be repaired efficiently by homologous recombination with a sister chromosome.
We speculate that the replication-independent DSBs are not the primary cause of cell death. We base this suggestion on the results in showing that growth rate of the cells affects the degree of cytotoxicity. The level of replication-independent DSBs should be the same in cells growing fast or slow but replication fork collapse is expected to be much more frequent in fast-growing than slow-growing cells. This model would be in agreement with analogous results obtained with base analogues and cisplatin which do require DNA replication for MMR-mediated cell death of dam
mutants. Using these agents as a precedent, DNA replication of O6
-meG/C to form either O6
-meG/C or O6
-meG/T in MNNG-treated dam
mutants, would provoke futile cycling where neither of these base pairs is perceived as being correct by the MMR system and leads to repeated rounds of repair [12
]. During this time, a replication fork encounters this gap and collapses as shown for cisplatin in and homologous recombination is required to restore the fork (). This scenario is supported by the data in , where the doubling time of the culture (and hence the number of replication forks) has a large effect on cytotoxicity. This same model can be applied to 2-aminopurine (2-AP) where 2AP/T and 2AP/C base pairs are possible. It has not been possible to test the model with 2-AP, however, due to massive chromosomal DNA breakdown presumably initiating at sites of DSBs [37
]. No such DNA degradation is observed in MNNG or cisplatin-treated dam
In the models above, homologous recombination (HR) is essential to restore integrity of the chromosome after methylation damage. It has been shown recently that HR in E. coli
is indeed required for resistance to methylating agents (MNNG and MMS) and its contribution is of the same magnitude as BER [25
]. The genetic data indicated that both RecF (repair of single-strand gaps) and RecBCD (repair of DSBs) pathways are necessary to prevent against toxicity with the latter being more important. AP-endonuclease-induced nicks at abasic sites of methylation damage leading to the formation of DSBs were predicted to be a major substrate for repair by HR.
In mammalian cells, inactivation of MMR results in tolerance to MNNG while MMR-proficiency leads to sensitivity, a phenomenon similar to that observed with an E. coli dam
]. Futile cycling by MMR at O6
-meG base pairs was also proposed as a model [40
] and recently data were obtained in vitro that supports this concept [42
]. It is not inconceivable that events similar to those in E. coli dam
mutants could occur in mammalian cells by the generation of a stable MMR-induced single-strand gap as the first step and replication fork collapse as the second followed by signaling of the apoptotic response. Recent results with Saccharomyces cerevisiae
exposed to MNNG indicated an MMR-dependent killing mechanism requiring HR for cell survival which is most likely mechanistically related to those in mammalian and E. coli dam
]. In contrast, sensitization by MMR in mammalian cells exposed to cisplatin is now in doubt [44
] which may reflect the difference between the action of these agents as shown here.