Cytotoxic and mutagenic properties of O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli cells

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 -alkyl-2'-deoxyguanosine ( -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 or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven -alkyl-dG lesions, with the alkyl group being Me, Et,Pr, Pr,Bu, Bu, orBu, and examined the impact of these lesions on DNA replication in cells. We found that replication past all the -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 -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 -alkyl-dG lesions and their recognition by the 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.

DNA replication studies of N-nitroso compound–induced O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli

“…The roles of SOS-induced DNA polymerases in bypassing the O 6 -POB-dG and O 6 -PHB-dG were also investigated by performing the assay in E. coli strains deficient in Pol II, Pol IV, Pol V, or all three in combination. It turned out that a substantial diminution in bypass efficiency was observed only when all three SOS-induced DNA polymerases were depleted, which is consistent with the observations made previously for other O 6 -alkyl-dG lesions (30). No significant differences were observed for the bypass efficiencies of O 6 -POB-dG and O 6 -PHB-dG lesions in AB1157 cells and the isogenic cells with a single depletion of any of the three polymerases (Fig.…”

“…The native PAGE analysis also facilitated the quantification of the mutation frequencies of O 6 -POB-dG and O 6 -PHB-dG. In agreement with previous findings (30), the major type of mutation was G 3 A transition for both O 6 -POB-dG and O 6 -PHB-dG in all E. coli strains (Fig. 5B).…”

“…Furthermore, O 6 -CM-dG could be induced in cultured human cells upon treatment with azaser- ine, a pancreatic carcinogen that can be converted to diazoacetate by cellular esterases (25). Multiple lines of evidence suggested that the O 6 position of 2Ј-deoxyguanosine is the most facile exocyclic atom to be alkylated on nucleobases (26), and the resulting lesions alter the hydrogen bonding property of the guanine base (6,(27)(28)(29)(30). In this vein, O 6 -methyl-dG (O 6 -Me-dG) and O 6 -POB-dG have been implicated in lung tumorigenesis mediated by NNK (12,(31)(32)(33), and O 6 -CM-dG may contribute to the mutations found in TP53 tumor suppressor gene of human gastrointestinal tumors (19) and is correlated with colorectal cancer (6).…”

Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells

“…Many exogenous and endogenous agents can react with DNA to induce DNA damage, and alkylation constitutes a major and diverse type of DNA damage (2). Among the four exocyclic oxygen atoms of nucleobases, the O 6 position of guanine is the most facile site to be alkylated (3). Appreciable levels of O 6 -alkyl-2Ј-deoxyguanosine (O 6 -alkyl-dG) 2 could be detected in human tissues (4,5).…”

“…dG), strongly impede DNA replication (29). Unrepaired O 6 -alkyl-dG lesions also display strong miscoding potential during DNA replication and induce almost exclusively the G3 A transition mutation (3,26). Unlike the extensively studied O 6 -Me-dG and O 6 -Et-dG, the biological effects of bulkier O 6 -alkyl-dG lesions are less well understood, despite the fact that the latter lesions are detectable in tissue DNA of animals after exposure to N-nitroso compounds (30 -32).…”