Kinetic Analysis of Translesion Synthesis Opposite Bulky N- and O-Alkylguanine DNA Adducts by Human DNA

REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addition to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N2-alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH2(6-benzo[a]pyrenyl) (BP) andO6-alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O6-PobG, which caused almost complete blockage. Steadystate kinetic parameters (kcat/Km) were similar for insertion of dCTP opposite G and N2-G adducts but were severely reduced opposite the O6-G adducts. REV1 showed apparent pre-steadystate burst kinetics for dCTP incorporation only opposite N2-BPG and little, if any, opposite G, N2-benzyl (Bz)G, or O6-BzG. The maximal polymerization rate (kpol 0.9 s 1) opposite N2-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 boundN2-BPG-adductedDNA3-foldmore tightly thanunmodified G-containing DNA. These results and the lack of an elemental effect ((Sp)-2 -deoxycytidine 5 -O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N2-BPG.Weconclude that humanREV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N2-adduct at G but not to O6-adducts.