In vitro bypass of the major malondialdehyde- and base propenal-derived DNA adduct by human Y-family DNA polymerases κ, ι, and Rev1.
3-(2'-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M(1)dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M(1)dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M(1)dG → A and M(1)dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M(1)dG in vivo. Previous reports described the bypass of M(1)dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M(1)dG by the human Y-family DNA polymerases κ, ι, and Rev1. M(1)dG was incorporated into template-primers containing either dC or dT residues 5' to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol ι, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M(1)dG:dC > M(1)dG:dG > M(1)dG:dT ∼ M(1)dG:dA, but neither hPol ι nor Rev1 extended M(1)dG-containing template-primers. Liquid chromatography-mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3'-GXC-5' template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M(1)dG in the 3'-GXT-5' template sequence. The results indicate that DNA hPol κ or the combined action of hPol ι or Rev1 and hPol κ bypass M(1)dG residues in DNA and generate products that are consistent with some of the mutations induced by M(1)dG in mammalian cells.
Maddukuri
Leena
L
A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Eoff
Robert L
RL
Choi
Jeong-Yun
JY
Rizzo
Carmelo J
CJ
Guengerich
F Peter
FP
Marnett
Lawrence J
LJ
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008315:Malondialdehyde / Q000737:chemistry / Q000378:metabolism; D009153:Mutagens; D010545:Peroxides; D000099270:Y-Family DNA Polymerases
D004247:DNA; D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D008315:Malondialdehyde; D009153:Mutagens; D010545:Peroxides; C057755:DNA hydroperoxide; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein