Abstract
Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G → A mutation, the frequency of which increased with the size of the alkyl group on the DNA. This could be attributed to the varied repair efficiencies of these lesions by O6-alkylguanine DNA alkyltransferase (MGMT) in cells, which involve the MGMT Ogt and, to a lesser extent, Ada. In conclusion, our study provides important new knowledge about the repair of the O6-alkyl-dG lesions and their recognition by the E. coli DNA replication machinery. Our results suggest that the lesions' carcinogenic potentials may be attributed, at least in part, to their strong mutagenic potential and their efficient bypass by the DNA replication machinery.
Highlights
Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone
We first synthesized a series of ODNs harboring site- incorporated O6-alkylguanine lesions (Fig. 1) and confirmed their identities by using electrospray ionization (ESI)–MS and MS/MS analyses
We assessed how the replicative bypass of these lesions is influenced by translesion synthesis DNA polymerases and two DNA repair proteins, i.e. Ada and Ogt
Summary
Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. O6-alkyl-2-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. We found that replication past all the O6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. These lesions directed exclusively the G 3 A mutation, the frequency of which increased with the size of the alkyl group on the DNA. It is important to understand how the O6-alkyl-dG lesions perturb the efficiency and accuracy of DNA replication and how they are repaired
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