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. 2014 Jul;35(7):1461-8.
doi: 10.1093/carcin/bgu003. Epub 2014 Jan 7.

Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct

Ying-Chih Lin  1 , Liang Li  2 , Alena V Makarova  3 , Peter M Burgers  3 , Michael P Stone  2 , R Stephen Lloyd  4
Affiliations

Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct

Ying-Chih Lin et al. Carcinogenesis. 2014 Jul.

Abstract

Aflatoxin B1 (AFB1) is a known carcinogen associated with early-onset hepatocellular carcinoma (HCC) and is thought to contribute to over half a million new HCCs per year. Although some of the fundamental risk factors are established, the molecular basis of AFB1-induced mutagenesis in primate cells has not been rigorously investigated. To gain insights into genome instability that is produced as a result of replicating DNAs containing AFB1 adducts, site-specific mutagenesis assays were used to establish the mutagenic potential of the persistent ring-opened AFB1 adduct, AFB1-formamidopyrimidine (AFB1-FAPY). This lesion was highly mutagenic, yielding replication error frequencies of 97%, with the predominant base substitution being a G to T transversion. This transversion is consistent with previous mutational data derived from aflatoxin-associated HCCs. In vitro translesion synthesis assays demonstrated that polymerase (pol) ζ was the most likely candidate polymerase that is responsible for the G to T mutations induced by this adduct.

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Figures

Fig. 1.
Fig. 1.
(A) Formation of the AFB1-DNA adducts. AFB1 is metabolically activated to AFB1-8,9-epoxide, which interacts with deoxyguanosine to form the cationic AFB1-N7-Gua; further hydrolysis gives rise to AFB1-FAPY. (B) Replication blockage of pol δ by AFB1-FAPY. −10, oligodeoxynucleotide primers were annealed to ND or AFB1-FAPY-containing DNA templates. Primer extensions were catalyzed by 1nM (lane 2, 5) or 50nM (lane 3, 6) pol δ in the presence of 100 μM dNTPs. G*, adducted site.
Fig. 2.
Fig. 2.
Replication bypass of AFB1-FAPY by yeast pol ζ. The −1 or 0 oligodeoxynucleotide primers with different 3′ end (where N represents either A, C, G or T) were annealed with ND or AFB1-FAPY-adducted DNA templates. (A) Single-nucleotide incorporations and primer extension reactions were catalyzed by 5nM (ND) or 50nM (AFB1-FAPY) pol ζ in the presence of 100 μM individual or all dNTPs. (B) Oligodeoxynucleotide primer extensions from the matched C or mismatched A, G and T 3′ terminus opposite ND or AFB1-FAPY were catalyzed by 10nM pol ζ in the presence of 100 μM dNTPs.
Fig. 3.
Fig. 3.
Replication bypass of AFB1-FAPY by human pol κ. Oligodeoxynucleotide primers (−10, −1 or 0) with different 3′ termini (designated as N, which represents either A, C, G or T) were annealed to ND or AFB1-FAPY-containing DNA templates. (A) Primer extension reactions were catalyzed by increasing concentrations of pol κ (2 or 10nM) in the presence of 100 μM dNTPs. (B) Single-nucleotide incorporation and primer extension reactions were conducted by 2nM (ND) or 10nM (AFB1-FAPY) pol κ in the presence of 20 μM (ND) or 100 μM (AFB1-FAPY) combined or individual dNTPs. (C) Primer extensions from the matched C or mismatched A, G, and T 3′ terminus opposite AFB1-FAPY or ND were catalyzed by 10 or 0.5nM pol κ in the presence of 20 μM dNTPs. G*, indicates the position of the adducted site.
Fig. 4.
Fig. 4.
Resumption of replication by pol δ downstream of AFB1-FAPY. +2, +3 or +5 oligodeoxynucleotide primers with either matched C or mismatched A opposite the lesion or control site were annealed to ND or AFB1-FAPY-containing DNA templates. (A and B) Primer extensions were catalyzed on matched and mismatched primers, respectively. All reactions were catalyzed by 50nM pol δ in the presence of 100 μM dNTPs. G*, adducted site.
Fig. 5.
Fig. 5.
Proposed model of TLS past AFB1-FAPY. The mutagenic bypass of AFB1-FAPY by pol ζ. The current model suggests that pol δ can synthesize up to one nucleotide prior to the lesion, followed by a polymerase switch to pol ζ. Pol ζ is proposed to preferentially insert an A opposite the lesion and extend synthesis several nucleotides downstream from the inserted A (indicated by dark green lines). This would be followed by a second polymerase switch to pol δ that catalyzes resumption of normal DNA replication.
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