Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase η

The N2 position of guanine (G) is one of the major sites for DNA modification by various carcinogens. Eight oligonucleotides with varying adduct bulk at guanine N2 were analyzed for catalytic efficiency and fidelity with human DNA polymerase (pol) eta, which is involved in translesion synthesis (TLS). Pol eta effectively bypassed N-2-methyl(Me)G, N-2-ethyl(Et)G, N-2-isobutyl(Ib)G, N-2-benzyl(Bz)G, and N-2-CH2(2-naphthyl)G but was severely blocked at N-2-CH2(9-anthracenyl)G (N-2-AnthG) and N-2-CH2(6-benzo[a]pyrenyl)G (N-2-BPG). Steady-state kinetic analysis showed proportional decreases of k(cat)/K-m in dCTP insertion opposite N-2-AnthG and N-2-BPG (73 and 320-fold) and also k(cat)/K-m in next-base extension from a C paired with each adduct (15 and 51-fold relative to G). Frequencies of dATP misinsertion and extension beyond mispairs were also proportionally increased (70 and 450-fold; 12 and 44-fold) with N-2-AnthG and N-2-BPG, indicating the effect of adduct bulk on blocking and misincorporation in TLS by pol eta. N-2-AnthG and N-2-BPG also greatly decreased the pre-steady-state kinetic burst rate (25 and 125-fold) compared to unmodified G. N-2-AnthG decreased dCTP binding affinity (2.6-fold) and increased DNA substrate binding affinity. These results and the small kinetic thio effects (S-p-dCTP alpha S) suggest that the early steps, possibly conformational change, are interfered with by the bulky adducts. In contrast, human pol delta bypassed adducts effectively up to N-2-EtG but was strongly blocked by N-2-IbG and larger adducts. We conclude that TLS DNA polymerases may be required for the efficient bypass of pol delta-blocking N-2-G adducts bulkier than N-2-EtG in human cells, and the bulk size can be a major factor for efficient and error-free bypass at these adducts by TLS DNA polymerases. (c) 2005 Elsevier Ltd. All rights reserved.