Mutagenic Replication of N²-Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV

Abstract

Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N2-position of guanine to produce N2-BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2′-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]-guanosine and 2′-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2′-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson–Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N2-BP-dG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N2-BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.

Active Site Interactions Impact Phosphoryl Transfer during Replication of Damaged and Undamaged DNA by Escherichia coli DNA Polymerase I

“…Molecular modeling experiments provide insight for the possible reactive conformations with T7 pol and BF. − In these structures, the template N 2 -BP-G is in the syn -conformation, with BP-moiety pointing toward the major groove and the Hoogsteen hydrogen bonding face pointing toward the incoming dNTP. Our recent results support this hydrogen-bonding interaction . In these models, the Arg is predicted to be in position to make the hydrogen-bonding interactions between the primer terminus and dNTP …”

“…Our recent results support this hydrogen-bonding interaction. 47 In these models, the Arg is predicted to be in position to make the hydrogen-bonding interactions between the primer terminus and dNTP. 80 The dNTP concentration dependence on k pol is shown in Figure S10−S13.…”

“…The phosphoramidites for O 6 mG, and oxoG were purchased from Glen Research (Sterling, VA). The oligodeoxy­nucleotides containing 6-amino-1-(2-deoxy-β- d - erythro -pentofuranosyl)-1,5-dihydro-4 H -imidazo­[4,5- c ]­pyridine-4-one (3DG), O 2 -POB-T, and N 2 -BP-dG were synthesized as described. − The sequence of the template strand was 5′-CTG CGA CAX CTG CGT CTG CGG TGC-3′ with X being A,C,G,T, O 6 mG, oxoG, N 2 -BP-G, and O 2 -POB-T. The primer strand was 5′-GCA CCG CAG ACX-3′ with X being G or 3DG.…”

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine

“…Our study suggests that PCNA123 mediates the complementing activities of PolY and PolB1 not only upon encountering the lesion, but also after the lesion has been bypassed. In response to other DNA lesions, the kinetics of lesion bypass by PolY have already been characterized (44)(45)(46)(47)(48)(49). In some of the previous analyses, 'pause sites' were noted during PolY lesion bypass, indicating points of inefficient catalysis and extension from these lesions.…”