Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua

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.

Keywords: DNA Polymerase; DNA Replication; Environmental Carcinogenesis; Genomic Instability; Hepatocellular Carcinoma; Site-directed Mutagenesis; Site-specific DNA Adduct; Translesion DNA Synthesis.

FIGURE 1.

Chemical structures of the AFB₁-DNA adducts, adapted from Ref. . CYP, cytochrome P450.

FIGURE 2.

Replication bypass of AFB₁-N7-Gua by pol δ. The −10, −1, or 0 primers with either C or A at the 3′ end were annealed to ND or AFB₁-N7-Gua adducted DNA templates. A, primer extensions were catalyzed by 1 nm (lanes 2 and 5) or 50 nm (lanes 3 and 6) pol δ in the presence of 100 μm dNTPs. G*, adducted site. B, single-nucleotide incorporations and primer extensions were conducted by 50 nm pol δ in the presence of combined or individual dNTPs at a final concentration of 100 μm. C, dNTP combination and primer extension reactions were catalyzed by 50 nm pol δ in the presence of different combinations of 100 μm individual dNTPs. D, primer extensions from the 3′ end of the 0 primer with a matched C or mismatched A annealed opposite ND or AFB₁-N7-Gua were catalyzed by 50 nm pol δ in the presence of 100 μm dNTPs in a buffer containing 40 mm Hepes-KOH, pH 6.8, 10% glycerol, 0.2 mg/ml bovine serum albumin, 1 mm DTT, and 8 mm MgCl₂ for 1 h at 37 °C. E, single-nucleotide insertion opposite AFB₁-N7-Gua by pol δ-Exo. Primer extension reactions were catalyzed by 20 nm pol δ-Exo in the presence of 100 μm individual or all dNTPs.

FIGURE 3.

Single-nucleotide incorporations and primer extensions opposite AFB₁-N7-Gua by yeast pol ζ₄ and human pol κ, η, and ι. The −1 primer was annealed to DNA templates containing ND or AFB₁-N7-Gua. A–D, reactions were catalyzed by 10 nm pol ζ₄, 2 nm (ND) and 10 nm (AFB₁-N7-Gua) pol κ, 2 nm pol η catalytic core, and 1 nm pol ι, respectively. 100 μm individual or all dNTPs was used in panels A and B (AFB₁-N7-Gua) and D, whereas 20 μm was added in panels C and B (ND). G*, adducted site.

FIGURE 4.

Primer extensions past AFB₁-N7-Gua catalyzed by yeast pol ζ₄ and human pol κ, η, and ι. Four 0 primers with different 3′ ends (where N represents A, C, G, or T) were annealed to ND or AFB₁-N7-Gua adducted DNA templates. A, control reactions contained no polymerase. B–E, reactions were catalyzed by 5 nm pol ζ₄, 1 nm pol κ, 0.5 nm pol η, and 10 nm pol ι, respectively. Reactions in panels A, B, C, and E were conducted in the presence of 100 μm dNTPs, whereas 20 μm was used in panel D.

FIGURE 5.

Resumption of replication by pol δ downstream of AFB₁-N7-Gua. +2, +3, or +5 primers with either matched C or mismatched A opposite the lesion or control site were annealed to ND or AFB₁-N7-Gua-containing DNA templates. A and B, primer extensions were catalyzed on matched and mismatched primers, respectively. All reactions were catalyzed by 50 nm pol δ in the presence of 100 μm dNTPs. G*, adducted site.

FIGURE 6.

Proposed model of TLS past AFB₁-N7-Gua. The accurate and error-prone bypasses of AFB₁-N7-Gua by pol δ or ζ₄ and pol κ, respectively, are shown. For this lesion, the model proposes that pol δ can replicate past the lesion by correct insertion and extension from the damage site. Alternatively, if pol δ is blocked and dissociates one nucleotide prior to the adduct, pol ζ₄ can preferentially insert a correct base opposite and extend from the lesion (green lines). The mutagenic pathway involves the blocking of pol δ one nucleotide prior to the lesion followed by recruitment of pol κ to catalyze the insertion step and the more efficient extension from a mispaired A:G terminus (blue lines). Efficient resumption of normal replication by pol δ after the second polymerase switch is proposed to occur at least five nucleotides downstream of the mismatched site.