Inhibition of DNA replication fork progression and mutagenic potential of 1, N⁶-ethenoadenine and 8-oxoguanine in human cell extracts

• Published in: Nucleic acids research 2008-03, Vol.36 (4), p.1300-1308
• Main Authors: Tolentino, Joel H, Burke, Tom J, Mukhopadhyay, Suparna, McGregor, W. Glenn, Basu, Ashis K
• Format: Article
• Language: English
• Subjects: Adenine - analogs & derivatives; Adenine - metabolism; Cell Extracts; Cell Line; DNA Damage; DNA Replication; DNA-Directed DNA Polymerase - metabolism; Electrophoresis, Gel, Two-Dimensional; Genetic Vectors; Guanine - analogs & derivatives; Guanine - metabolism; HeLa Cells; Humans; Molecular Biology; Mutagenesis
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkm1157

Comparative mutagenesis of 1,N⁶-ethenoadenine (εA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past εA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, εA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G[rightward arrow]T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of εA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the εA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, εA[rightward arrow]T mutations increased 3-fold in products derived from the mismatched εA: G construct compared with those derived from the εA: T construct. More efficient repair of εA in the εA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol η have shown that this polymerase is highly error-prone when bypassing εA. To examine if pol η is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The εA: T construct induced εA[rightward arrow]G and εA[rightward arrow]C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, εA[rightward arrow]T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol η-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.