Kinetic analysis of translesion synthesis opposite bulky N2- and O6-alkylguanine DNA adducts by human DNA polymerase REV1.

Abstract:

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, N(2)-alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH(2)(6-benzo[a]pyrenyl) (BP) and O(6)-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 O(6)-PobG, which caused almost complete blockage. Steady-state kinetic parameters (k(cat)/K(m)) were similar for insertion of dCTP opposite G and N(2)-G adducts but were severely reduced opposite the O(6)-G adducts. REV1 showed apparent pre-steady-state burst kinetics for dCTP incorporation only opposite N(2)-BPG and little, if any, opposite G, N(2)-benzyl (Bz)G, or O(6)-BzG. The maximal polymerization rate (k(pol) 0.9 s(-1)) opposite N(2)-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N(2)-BPG-adducted DNA 3-fold more tightly than unmodified G-containing DNA. These results and the lack of an elemental effect ((S(p))-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N(2)-BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N(2)-adduct at G but not to O(6)-adducts.

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