PDBsum entry 2c22

Polymerase

PDB id: 2c22

Contents

Protein chain

342 a.a.

DNA/RNA

Ligands

DGT

Metals

_CA ×3

Waters ×110

References listed in PDB file

Key reference

Title

Efficient and high fidelity incorporation of dctp opposite 7,8-Dihydro-8-Oxodeoxyguanosine by sulfolobus solfataricus DNA polymerase dpo4.

Authors

H.Zang, A.Irimia, J.Y.Choi, K.C.Angel, L.V.Loukachevitch, M.Egli, F.P.Guengerich.

Ref.

J Biol Chem, 2006, 281, 2358-2372. [DOI no: 10.1074/jbc.M510889200]

PubMed id

16306039

Abstract

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.

Figure 1.
Extension of primers by Dpo4. Extension of a ^32P-labeled primer (13-mer, oligomer 1 of Scheme 1) opposite G or 8-oxoG (oligomer 2 of Scheme 1) was analyzed with increasing reaction times, as indicated by the gradient bars (0, 10, 30, 60, 90, 120, and 180 min, respectively).

Figure 8.
Quality of the electron density and DNA duplex conformations at the active sites in the ternary complexes. A, Dpo4-dG; B, Dpo4-dA; C, Dpo4-dC; D, Dpo4-ddG; E, Dpo4-ddC. The views are into the major groove, Fourier (3F[o] - 2F[c]) sum electron density is drawn at the 1σ level, Ca^2+ ions are yellow spheres, and phosphorus atoms of the α-, β-, and γ-phosphate groups of (d)dNTPs are highlighted in cyan, gray, and white, respectively.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 2358-2372) copyright 2006.