2j6t

From Proteopedia

Revision as of 08:42, 3 February 2008

TERNARY COMPLEX OF SULFOLOBUS SOLFATARICUS DPO4 DNA POLYMERASE, O6-METHYLGUANINE MODIFIED DNA, AND DATP.

Overview

We examined the effect of a single O6-methylguanine (O6-MeG) template, residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus, solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products, revealed that the enzyme accurately bypasses O6-MeG, with C being the, major product (approximately 70%) and T or A being the minor species, (approximately 20% or approximately 10%, respectively), consistent with, steady-state kinetic parameters. Transient-state kinetic experiments, revealed that kpol, the maximum forward rate constant describing, polymerization, for dCTP incorporation opposite O6-MeG was approximately, 6-fold slower than observed for unmodified G, and no measurable product, was observed for dTTP incorporation in the pre-steady state. The lack of, any structural information regarding how O6-MeG paired in a polymerase, active site led us to perform x-ray crystallographic studies, which show, that "wobble" pairing occurs between C and O6-MeG. A structure containing, T opposite O6-MeG was solved, but much of the ribose and pyrimidine base, density was disordered, in accordance with a much higher Km,dTTP that, drives the difference in efficiency between C and T incorporation. The, more stabilized C:O6-MeG pairing reinforces the importance of hydrogen, bonding with respect to nucleotide selection within a geometrically, tolerant polymerase active site.

About this Structure

2J6T is a Single protein structure of sequence from Sulfolobus solfataricus with and as ligands. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Known structural/functional Site: . Full crystallographic information is available from OCA.

Reference

Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by "wobble" pairing between O6-methylguanine and cytosine, but accurate bypass is preferred., Eoff RL, Irimia A, Egli M, Guengerich FP, J Biol Chem. 2007 Jan 12;282(2):1456-67. Epub 2006 Nov 14. PMID:17105728

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