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DNA replication studies of N-nitroso compound-induced O 6-alkyl-2'-deoxyguanosine lesions in Escherichia coli.

ABSTRACT: N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we assessed the cytotoxic and mutagenic properties of three O 6-alkyl-2'-deoxyguanosine (O 6-alkyl-dG) adducts, i.e. O 6-pyridyloxobutyl-dG (O 6-POB-dG) and O 6-pyridylhydroxybutyl-dG (O 6-PHB-dG), derived from tobacco-specific nitrosamines, and O 6-carboxymethyl-dG (O 6-CM-dG), induced by endogenous N-nitroso compounds. We also investigated two neutral analogs of O 6-CM-dG, i.e. O 6-aminocarbonylmethyl-dG (O 6-ACM-dG) and O 6-hydroxyethyl-dG (O 6-HOEt-dG). We found that, in Escherichia coli cells, these lesions mildly (O 6-POB-dG), moderately (O 6-PHB-dG), or strongly (O 6-CM-dG, O 6-ACM-dG, and O 6-HOEt-dG) impede DNA replication. The strong blockage effects of the last three lesions were attributable to the presence of hydrogen-bonding donor(s) located on the alkyl functionality of these lesions. Except for O 6-POB-dG, which also induced a low frequency of G ? T transversions, all other lesions exclusively stimulated G ? A transitions. SOS-induced DNA polymerases played redundant roles in bypassing all the O 6-alkyl-dG lesions investigated. DNA polymerase IV (Pol IV) and Pol V, however, were uniquely required for inducing the G ? A transition for O 6-CM-dG exposure. Together, our study expands our knowledge about the recognition of important NOC-derived O 6-alkyl-dG lesions by the E. coli DNA replication machinery.

SUBMITTER: Wang P

PROVIDER: S-EPMC6422096 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Wang Pengcheng P Leng Jiapeng J Wang Yinsheng Y

The Journal of biological chemistry 20190117 11

N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we ass ...[more]

PMID: 30655287

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Project description:DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O6-dG-derived ICLs. Although less abundant, O6-alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O6-2'-deoxyguanosine-butylene-O6-2'-deoxyguanosine (O6-dG-C4-O6-dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O6-dG-C4-O6-dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol ? is completely inhibited by O6-dG-C4-O6-dG ICLs. Moderate bypass activities are observed for human pol ? and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol ? exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol ? in O6-dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol ?. Together, this study provides the first set of biochemical evidence for the mutagenicity of O6-dG-derived ICLs.

Dataset Information

DNA replication studies of N-nitroso compound-induced O 6-alkyl-2'-deoxyguanosine lesions in Escherichia coli.

Publications

DNA replication studies of <i>N</i>-nitroso compound-induced <i>O</i><sup>6</sup>-alkyl-2'-deoxyguanosine lesions in <i>Escherichia coli</i>.

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