Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι

DNA polymerase (pol) iota 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 iota. Pol iota effectively bypassed N-2-methyl (Me) G and N-2-ethyl(Et)G, partially bypassed N-2-isobutyl(Ib)G and N-2-benzylG, and was blocked at N-2-CH2(2-naphthyl)G (N-2-NaphG), N-2-CH2(9-anthracenyl)G (N-2-AnthG), and N-2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of k(cat)/K-m for dCTP insertion opposite N-2-G adducts according to size, with a maximal decrease opposite N-2-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. N-2-IbG, N-2-NaphG, and N-2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (Sp-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N-2-EtG and N-2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N-2, N-2-diMeG compared with N-2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N-2-G adducts in cells.