Kinetic Evidence for Inefficient and Error-prone Bypass across Bulky N²-Guanine DNA Adducts by Human DNA Polymerase ι*

DNA polymerase (pol) ι has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol ι. Pol ι effectively bypassed N²-methyl (Me)G and N²-ethyl(Et)G, partially bypassed N²-isobutyl(Ib)G and N²-benzylG, and was blocked at N²-CH₂(2-naphthyl)G (N²-NaphG), N²-CH₂(9-anthracenyl)G (N²-AnthG), and N²-CH₂(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of k_cat/K_m for dCTP insertion opposite N²-G adducts according to size, with a maximal decrease opposite N²-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3–11-fold opposite adducts (highest with N²-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N²-IbG, N²-NaphG, and N²-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S_p-2′-deoxycytidine 5′-O-(1-thiotriphosphate)) opposite N²-EtG and N²-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N²,N²-diMeG compared with N²-EtG by pol η but not by pol ι is consistent with Hoogsteen base pairing by pol ι. Thus, polymerization by pol ι is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol ι may play a limited role in translesion synthesis on bulky N²-G adducts in cells.