Basis of miscoding of the DNA adduct N2,3-ethenoguanine by human Y-family DNA polymerases.
N(2),3-Ethenoguanine (N(2),3-εG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2'-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2'-fluoro-N(2),3-ε-2'-deoxyarabinoguanosine to investigate the miscoding potential of N(2),3-εG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N(2),3-εG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N(2),3-εG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N(2),3-εG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N(2),3-εG:dCTP base pair, whereas only one appears to be present in the case of the N(2),3-εG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N(2),3-εG.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Linlin
L
Zhao
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Matthew G
MG
Pence
Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Plamen P
PP
Christov
Northwestern University Synchrotron Research Center, Life Sciences Collaborative Access Team, Argonne, Illinois 60439.
Zdzislaw
Z
Wawrzak
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Gyeonggi-do 440-746, Republic of Korea.
Jeong-Yun
JY
Choi
Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Carmelo J
CJ
Rizzo
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Martin
M
Egli
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146. Electronic address: f.guengerich@vanderbilt.edu.
F Peter
FP
Guengerich
D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D018736:DNA Adducts; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; C015196:N(2),3-ethenoguanine; C415367:DNA polymerase beta2; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C404302:POLK protein, human; C546205:POLN protein, human