The efficiency to which an O6
mG lesion is repaired in E. coli
depends on the number of MTase molecules present in the cell and the affinities of those MTases for the lesion. During exponential growth, approximately 30 molecules of Ogt [15
] and 1 molecule of Ada [38
] are present. On this basis, we expected to observe a more dramatic decrease in repair in cells lacking Ogt (C216) as compared to the cells lacking Ada (C217). Data for the parental strains verify that the repair efficiency of the C216 strain is indeed lower than for the C217 strain. However, the affinity of O6
mG for Ada is greater than that for Ogt [39
], and therefore the difference in repair observed for the ada
mutants reflects multiple parameters. We therefore decided to group the repair efficiencies according to the MTase background of the parent cell.
Interestingly, we observed data that were consistent with a small amount of O6
mG repair in the C218 double mutant strain bearing no MTase activity. We rationalized that this low level of repair signal was most likely due to a population of genome construct containing guanine in place of O6
mG. The synthetic route used to produce the 16-mer lesion-containing insert utilizes a deprotection step that was reported to convert small amounts of the lesion to guanine [40
]. Mass spectrometry was conducted on the lesion-containing 16-mer and confirmed the presence of a species corresponding to ~5% guanine at the lesion site (data not shown). This result was consistent with the basal level of “repair” signal in the MTase deficient cells.
Strains lacking dam
showed repair levels that were similar to their parental counterparts (), indicating that these MMR proteins do not play a role in affecting repair of O6
mG:T mismatches to G:C base pairs. These results likely reflect the property of these proteins to act at d(GATC) sequences that are remote from the mismatch [19
]; Dam and MutH would not be expected to be present at O6
mG:T sites and hence they would not be expected to influence MTase repair of the lesion in our system. It was a formal possibility that MMR could also effectuate repair of O6
mG:T mismatches to G:C base pairs by a mechanism whereby MMR occurred before MTase repair. We also view this possibility as unlikely. By this model, MMR would have to replace the mismatched thymine in the daughter strand with cytosine before MTase repair of O6
mG to guanine could follow. As indicated above, O6
mG is highly mutagenic as evidenced by the observation that thymine is placed opposite the lesion nearly 100% of the time [8
]. Therefore, the absence of contribution from Dam or MutH to the repair of O6
mG:T mismatches was expected. It should also be noted that the mutator phenotype, which is associated with dam−
], had no significant effect on MTase repair efficiency.
Strains lacking MutS and MutL displayed a decrease in MTase repair. These results were interesting as MutS can bind to O6
mG base pairs [10
] and can theoretically shield the lesion from MTase repair. Indeed, it could be postulated that MutS complexes should compete with MTases proteins for access to O6
mG base pairs. However, our data suggest otherwise. Strains bearing MutS and MutL proteins displayed a higher efficiency of O6
mG repair by MTases, suggesting these proteins cooperate with Ada and Ogt.
The mechanism by which the presence of MutS and MutL stimulates MTase repair of O6
mG would likely involve (i) the identification of the lesion by MutS/MutL followed by (ii) the dissociation of the complex from the mismatch to present and facilitate and (iii) downstream repair events (). MutL has been shown to assist MutS in mismatch presentation by stimulating the formation of an α-shaped DNA loop containing the mismatch [42
], and the observation that the mutL−
mutants displayed similar decreases in MTase repair is consistent with previous observations that mutating either gene results in an identical defect in mismatch recognition [43
]. The observation that dam−
mutants do not show decreased levels of MTase repair makes it highly unlikely that the effects seen from knocking out mutS
are the result of creating a mutator phenotype. Taken together, it therefore appears that a novel role of MutS and MutL mismatch repair proteins in assisting O6
mG repair by MTases has been uncovered.
Hypothetical mechanism for stimulation of MTase repair by VSPR proteins
The vsr− strains, in addition to mutS− and mutL− strains, show a significant decrease in MTase repair (, dark gray columns). We rationalize that Vsr dependent stimulation of one or both MTases () increases the likelihood that (after mismatch recognition by MutS and MutL) Vsr, and not MutH, initiates repair of G:T mismatches that result from MTase repair of O6mG:T base pairs. Subsequently, VSPR, and not MMR, corrects the mismatch to the native G:C pairing with high efficiency.
This model is attractive from a number of perspectives. First, VSPR is the most energetically advantageous mechanism by which to process O6mG:T mismatches to G:C base pairs. As described above, VSPR requires fewer protein components and characteristically replaces a much shorter segment of the error-containing DNA strand as compared to MMR. Second, it may be that the cell depends on VSPR for correction of G:T mispairs. Given that O6mG:T mismatches arise through replication past the lesion-containing DNA strand, MMR would be targeted to the DNA strand opposite the lesion and reiteratively regenerate a MMR substrate. Eventually, if futile attempts to repair the mismatch do not kill the cell, the DNA becomes fully methylated at the d(GATC) sites by Dam, thereby inactivating MMR. At this point, correct repair of O6mG:T mismatches depends on MTase repair followed by VSPR. Therefore, stimulation of MTase repair by VSPR proteins may promote cooperative repair, which ultimately becomes necessary. Third, it is possible that cooperation between the VSPR and MTase systems could have assisted cellular survival and influenced the evolution of E. coli. If these bacteria have been continually challenged with the task of correcting post-replicative O6mG:T mismatches, it is plausible that cooperation between the MTase and VSPR proteins developed over time. In doing so, the organism would have gained a survival advantage by networking repair pathways to correct a cytotoxic lesion with high efficiency. Finally, the model presented here, based upon observations made on viruses replicating in living cells, could be studied in parallel from a biochemical perspective to provide further mechanistic insight into cooperation among DNA repair pathways.