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Kinetic evidence for inefficient and error-prone bypass across bulky N 2-guanine DNA adducts by human DNA polymerase
- Kinetic evidence for inefficient and error-prone bypass across bulky N 2-guanine DNA adducts by human DNA polymerase
- Choi J.-Y.; Guengerich F.P.
- Ewha Authors
- Issue Date
- Journal Title
- Journal of Biological Chemistry
- Journal of Biological Chemistry vol. 281, no. 18, pp. 12315 - 12324
- SCIE; SCOPUS
- Document Type
- 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 N 2-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 kcat/Km 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 (Sp-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 N 2,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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
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