Abstract
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 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 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 (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 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 N2-G adducts in cells.
Highlights
Cell death, leading in turn to detrimental effects, including aging and cancer [3]
We have previously studied some of the details of blockage and misincorporation with the model replicative DNA polymerases pol T7Ϫ and HIV-1 RT in work focused on various modifications of G at the C8 (8-oxoG), O6 (O6-MeG and O6-BzG), and N2 atoms (N2-MeG, N2-EtG, N2-IbG, N2-BzG, N2-AnthG, N2-(2,3,4-trihydroxy-1-butyl)G, 8-hydroxy-1,N2-propanoG, 8-hydroxy-6-methyl-1,N2-propanoG, and the styrene oxide and BPDE products formed at the G N2 atom) of guanine [17, 31,32,33,34,35]
The effect of adduct size is consistently seen in one TLS polymerase, pol , pol is more resistant to bulky adducts than pol T7Ϫ and HIV-1 RT [18]
Summary
Materials—Unlabeled dNTPs were purchased from Amersham Biosciences. Sp-dCTP␣S and Rp-dCTP␣S were purchased from Biolog Life Science Institute (Bremen, Germany). [␥-32P]ATP (specific activity 3,000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Reactions were initiated by rapid mixing of 32Pprimer-template/polymerase mixtures (12.5 l) with the dNTP-Mg2ϩ complex (10.9 l) and quenched with 0.3 M EDTA after times varying from 5 ms to 4 s (or 30 s for N2-IbG-, N2-NaphG-, and N2AnthG-containing oligonucleotides). Phosphorothioate Analysis—With the 32P-primer annealed to either an unmodified or adducted template, reactions were initiated by rapid mixing of 32P-primer-template/polymerase mixtures (12.5 l) with Sp-dCTP␣S-Mg2ϩ complex (or dCTP-Mg2ϩ) (10.9 l) and quenched with 0.3 M EDTA after reaction times varying from 5 ms to 4 s (or 30 s for N2-AnthG-containing DNA). A graph of the burst rate (kobs) versus dCTP concentration was fit to the hyperbolic equation, kobs ϭ kpol[dNTP]/([dNTP] ϩ Kd), where kpol represents the maximal rate of nucleotide incorporation and dCTP d is the equilibrium dissociation constant for dCTP [41, 42]
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