2c22

From Proteopedia

Revision as of 15:50, 5 November 2007

EFFICIENT AND HIGH FIDELITY INCORPORATION OF DCTP OPPOSITE 7,8-DIHYDRO-8-OXODEOXYGUANOSINE BY SULFOLOBUS SOLFATARICUS DNA POLYMERASE DPO4

Overview

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.

About this Structure

2C22 is a Single protein structure of sequence from Sulfolobus solfataricus with CA and DGT as ligands. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Structure known Active Site: AC1. Full crystallographic information is available from OCA.

Reference

Efficient and high fidelity incorporation of dCTP opposite 7,8-dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA polymerase Dpo4., Zang H, Irimia A, Choi JY, Angel KC, Loukachevitch LV, Egli M, Guengerich FP, J Biol Chem. 2006 Jan 27;281(4):2358-72. Epub 2005 Nov 22. PMID:16306039

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