Error-prone Replication Bypass of the Primary Aflatoxin B1 DNA Adduct, AFB1-N7-Gua

Ying-Chih Lin,Liang Li,Alena V Makarova,Peter M Burgers,Michael P Stone,R Stephen Lloyd

doi:10.1074/jbc.m114.561563

Abstract

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths worldwide. The highest rates of early onset HCCs occur in geographical regions with high aflatoxin B1 (AFB1) exposure, concomitant with hepatitis B infection. Although the carcinogenic basis of AFB1 has been ascribed to its mutagenic effects, the mutagenic property of the primary AFB1-DNA adduct, AFB1-N7-Gua, in mammalian cells has not been studied extensively. Taking advantage of the ability to create vectors containing a site-specific DNA adduct, the mutagenic potential was determined in primate cells. This adduct was highly mutagenic following replication in COS-7 cells, with a mutation frequency of 45%. The spectrum of mutations was predominantly G to T base substitutions, a result that is consistent with previous mutation data derived from aflatoxin-associated HCCs. To assess which DNA polymerases (pol) might contribute to the mutational outcome, in vitro replication studies were performed. Unexpectedly, replicative pol δ and the error-prone translesion synthesis pol ζ were able to accurately bypass AFB1-N7-Gua. In contrast, replication bypass using pol κ was shown to occur with low fidelity and could account for the commonly detected G to T transversions.

Highlights

Aflatoxin B1 exposure causes mutations that are associated with liver cancer
Mutagenic Potential of aflatoxin B1 (AFB1)-N7-Gua Adducts in Primate Cells—To assess whether the AFB1-N7-Gua adducts (Fig. 1) are mutagenic precursors in mammalian cells, we generated an ss shuttle vector pMS2 containing site-specific AFB1-N7-Gua adduct or ND dG control; the vector was subsequently transfected into African green monkey kidney COS-7 cells
The second most common mutation type was G to A transitions (ϳ10%) followed by G to C transversions and deletions (Ͻ3%). These data indicate that AFB1-N7-Gua is a highly mutagenic precursor in primate cells, predominantly inducing a G to T transversion, which is consistent with observations of Hepatocellular carcinomas (HCCs) patient samples from areas experiencing high aflatoxin exposure [13, 14]

Summary

Background

Aflatoxin B1 exposure causes mutations that are associated with liver cancer. Results: The AFB1-N7-Gua adduct is highly mutagenic in primate cells, with contributions by both replicative and translesion synthesis DNA polymerases. AFB1-N7-Gua-induced Mutagenesis infection and aflatoxin exposure revealed that more than half of the HCC samples contained a G to T mutation at the third position in codon 249 (AGG) of the p53 tumor suppressor gene [13, 14] This mutation represents a biomarker of aflatoxin exposure as it is not detected in solely HBV-associated HCCs. Multiple experimental mutagenesis studies have shown that the predominant mutation caused by AFB1 exposure is G to T transversion [15,16,17,18]. The present study was designed to examine the mutagenic potential of AFB1-N7-Gua adducts in primate cells using a site-specific mutagenesis approach and subsequently test the potential role of eukaryotic DNA polymerases in replication bypass of this lesion by in vitro replication assays

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION