Biochemical Basis of Genotoxicity of Heterocyclic Arylamine Food Mutagens: HUMAN DNA POLYMERASE η SELECTIVELY PRODUCES A TWO-BASE DELETION IN COPYING THE N2-GUANYL ADDUCT OF 2-AMINO-3-METHYLIMIDAZO[4,5-f]QUINOLINE BUT NOT THE C8 ADDUCT AT THE NarI G3 SITE

Jeong-Yun Choi,James S Stover,Karen C Angel,Goutam Chowdhury,Carmelo J Rizzo,F Peter Guengerich

doi:10.1074/jbc.m605699200

Jeong-Yun Choi, James S Stover + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m605699200

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Abstract

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) eta extended primers beyond template G-IQ adducts better than did pol kappa and much better than pol iota or delta. In 1-base incorporation studies, pol eta inserted C and A, pol iota inserted T, and pol kappa inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol eta. Mass spectrometry of pol eta extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a -2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol eta products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol eta and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.

Highlights

The modification of DNA by chemical and physical agents is a common process and leads to loss of genetic stability [1], a factor in aging, cancer, and various other maladies, e.g. cardiovascular disease [2]
The results show that pol ␦ is completely blocked by the IQ adducts and that pol ␩ is the most efficient of the translesion DNA polymerases in copying. pol ␩ exhibited different behavior with both of the two IQ
Extension of Primers Opposite C8- and N2-IQ Adducts in Templates—Initial studies were done with all four dNTPs using each of the human DNA polymerases available (␦, ␩, ␫, and ␬)

Summary

Biochemical Basis of Genotoxicity of Heterocyclic Arylamine Food Mutagens

HUMAN DNA POLYMERASE ␩ SELECTIVELY PRODUCES A TWO-BASE DELETION IN COPYING THE N2-GUANYL ADDUCT OF 2-AMINO-3-METHYLIMIDAZO[4,5-f]QUINOLINE BUT NOT THE C8 ADDUCT AT THE NarI G3 SITE*□S. Using methods developed in our laboratories, we have been able to avoid producing mixtures of heterocyclic amine DNA adducts with the use of strategies for synthesis of appropriately modified monomers (24 –26) With this strategy, we were able to synthesize both of the major DNA adducts formed by IQ, the N 2- and C8-guanyl adducts (Fig. 1). Both of these adducts are formed upon reaction of DNA with N-hydroxy IQ [27], but the proportion of the N 2-IQ adduct increases with time in vivo (in rodents) due to much slower repair [27, 28] Both adducts were positioned at both the G1 and the G3 sites in the NarI sequence, which are known to be resistant and sensitive, respectively, to frameshift mutations with AAF [19, 29]. Both phosphodiesterase treatments confirmed the expected positions of the G1 and G3 site adducts in the synthetic templates containing C8- and N 2-IQ (data not presented)

Reaction Conditions for Enzyme

Km kcat

RESULTS

Theoretical fragmentation

DISCUSSION