2j6s: Difference between revisions
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==Overview== | ==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 .. | 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== | ==About this Structure== | ||
2J6S is a | 2J6S is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Sulfolobus_solfataricus Sulfolobus solfataricus] with CA and DTP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2J6S OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: translesion dna synthesis]] | [[Category: translesion dna synthesis]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 12:51:41 2007'' | ||
