Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases.
Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.
Yasui
Manabu
M
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.
Suzuki
Naomi
N
Miller
Holly
H
Matsuda
Tomonari
T
Matsui
Saburo
S
Shibutani
Shinya
S
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003853:Deoxyribonucleosides / Q000737:chemistry*; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides; D013698:Templates, Genetic
D018736:DNA Adducts; D003853:Deoxyribonucleosides; D009838:Oligodeoxyribonucleotides; C077801:2'-deoxyxanthosine; D009569:Nitric Oxide; D004259:DNA-Directed DNA Polymerase