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. 2009 Jan 16;284(3):1732-40.
doi: 10.1074/jbc.M807296200. Epub 2008 Nov 3.

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Matthew G Pence  1 , Patrick Blans, Charles N Zink, Thomas Hollis, James C Fishbein, Fred W Perrino
Affiliations

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Matthew G Pence et al. J Biol Chem. .

Abstract

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP VSports手机版. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration. .

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Figures

FIGURE 1.
FIGURE 1.
Extension opposite N2-ethyl-Gua or Gua by DNA Pol ι in the presence of MgCl2 or MnCl2. Primer extension assays were carried out as described under “Experimental Procedures.” The annealed primer (12-mer) and templates containing either N2-ethyl-Gua or Gua (A) were incubated with 10 nm DNA pol ι and increasing concentrations of MgCl2 (B) or MnCl2 (C). Maximal primer extension was observed at 2 mm MgCl2 and 0.075 mm MnCl2. In single nucleotide extension reactions with only dTTP or dCTP using the 14-mer primer, the 15-mer product of dTMP insertion migrates to the position of the upper band and the 15-mer product of dCMP insertion migrates to the position of the lower band (data not shown).
FIGURE 2.
FIGURE 2.
The structures of DNA pol ι·N2-ethyl-Gua Complexes with incoming dCTP or dTTP. A, the structure of DNA pol ι containing N2-ethyl-Gua and incoming dCTP shows the N2-ethyl-Gua base rotated into the syn configuration. B, the structure of DNA pol ι containing N2-ethyl-Gua and incoming dTTP shows the N2-ethyl-Gua base in the anti configuration and the N2-adduct protruding into the minor groove. C, electron density around the N2-ethyl adduct and incoming dCTP. D, electron density around the N2-ethyl adduct and the γ phosphate of the incoming dTTP.
FIGURE 3.
FIGURE 3.
Superposition of DNA pol ι complexes with anti and syn configurations of N2-ethyl-Gua and Gua. A, comparisons of the PAD domain in DNA pol ι with N2-ethyl-Gua (green) or Gua (PDB code 2ALZ, magenta) rotated into the syn configuration demonstrate the change in position of the backbone atoms of the PAD loop. B, the repositioning of the loop is not observed when comparing structures of DNA pol ι with N2-ethyl-Gua (blue) or Gua (PDB code 2FLP, yellow) rotated into the anti configuration.
FIGURE 4.
FIGURE 4.
Repositioning of Lys309 in the structure of DNA pol ι with N2-ethyl-Gua in the syn configuration. A, the Lys309 side chain in DNA pol ι in complex with N2-ethyl-Gua (green) in the syn configuration shifts ∼9Å relative to the position of Lys309 in DNA pol ι complexed with syn Gua (PDB code 2ALZ, magenta). B, with N2-ethyl-Gua in the anti conformation (blue) Lys309 remains in a similar position relative to the Lys309 in the DNA pol ι·Gua complex (PDB code 2FLP, yellow).
FIGURE 5.
FIGURE 5.
A N2-ethyl-Gua:Cyt wobble base in the active site of Y family DNA polymerases ι, κ, and η. A, Gln59 in DNA pol ι tightly coordinates the template base and sterically clashes with N2-ethyl-Gua when forming a wobble base pair with the incoming dCTP. B, DNA pol κ lacks a corresponding residue to Gln59 and accommodates the wobble base pair geometry. C, yeast DNA pol η has a Gln55 that corresponds to Gln59 in DNA pol ι but the more open active site accommodates the N2-ethyl-Gua:Cyt wobble base pair.
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