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, 19 (11), 1506-17

Translesion Synthesis Past the C8- And N2-deoxyguanosine Adducts of the Dietary Mutagen 2-Amino-3-methylimidazo[4,5-f]quinoline in the NarI Recognition Sequence by Prokaryotic DNA Polymerases

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Translesion Synthesis Past the C8- And N2-deoxyguanosine Adducts of the Dietary Mutagen 2-Amino-3-methylimidazo[4,5-f]quinoline in the NarI Recognition Sequence by Prokaryotic DNA Polymerases

James S Stover et al. Chem Res Toxicol.

Abstract

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is found in cooked meats and forms DNA adducts at the C8- and N2-positions of dGuo after appropriate activation. IQ is a potent inducer of frameshift mutations in bacteria and is carcinogenic in laboratory animals. We have incorporated both IQ-adducts into the G1- and G3-positions of the NarI recognition sequence (5'-G1G2CG3CC-3'), which is a hotspot for arylamine modification. The in vitro replication of the oligonucleotides was examined with Escherichia coli pol I Klenow fragment exo-, E. coli pol II exo-, and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and the extension products were sequenced by tandem mass spectrometry. Replication of the C8-adduct at the G3-position resulted in two-base deletions with all three polymerases, whereas error-free bypass and extension was observed at the G1-position. The N2-adduct was bypassed and extended by all three polymerases when positioned at the G1-position, and the error-free product was observed. The N2-adduct at the G3-position was more blocking and was bypassed and extended only by Dpo4 to produce an error-free product. These results indicate that the replication of the IQ-adducts of dGuo is strongly influenced by the local sequence and the regioisomer of the adduct. These results also suggest a possible role for pol II and IV in the error-prone bypass of the C8-IQ-adduct leading to frameshift mutations in reiterated sequences, whereas noniterated sequences result in error-free bypass.

Figures

Figure 1
Figure 1
Structures of IQ and its C8- and N2-dGuo-adducts.
Figure 2
Figure 2
Modified oligonucleotides (1 and 2) and primers used in extension reactions.
Figure 3
Figure 3
Full-length extension of the oligonucleotides 1 and 2 by Kf, pol II, and Dpo4.
Figure 4
Figure 4
LC-MS/MS analysis of the Kf extension products of oligonucleotide 1b. (A) TIC spectrum; (B) CID spectrum from m/z 946.7; and (C) CID spectrum from m/z 842.5.
Figure 5
Figure 5
LC-MS/MS analysis of the Kf extension products of oligonucleotide 2c. (A) TIC spectrum and (B) CID spectrum of m/z 1152.8.
Scheme 1
Scheme 1
LC-MS/MS Sequencing
Scheme 2
Scheme 2
One- and Two-Base Slippage Mechanism for pol II Bypass of the C8-IQ-Adduct in the Dinucleotide Repeat Position of the NarI Sequence (1b)
Scheme 3
Scheme 3
Mechanism for the Bypass and Extension of Modified Template 1b by Dpo4

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