2j6t
From Proteopedia
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==Overview== | ==Overview== | ||
| - | We examined the effect of a single O6-methylguanine (O6-MeG) template | + | 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== | ||
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[[Category: Sulfolobus solfataricus]] | [[Category: Sulfolobus solfataricus]] | ||
[[Category: Egli, M.]] | [[Category: Egli, M.]] | ||
| - | [[Category: Eoff, R | + | [[Category: Eoff, R L.]] |
| - | [[Category: Guengerich, F | + | [[Category: Guengerich, F P.]] |
[[Category: Irimia, A.]] | [[Category: Irimia, A.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
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[[Category: translesion dna synthesis]] | [[Category: translesion dna synthesis]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:59:50 2008'' |
Revision as of 15:59, 21 February 2008
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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
Page seeded by OCA on Thu Feb 21 17:59:50 2008
Categories: DNA-directed DNA polymerase | Single protein | Sulfolobus solfataricus | Egli, M. | Eoff, R L. | Guengerich, F P. | Irimia, A. | CA | DTP | Dna damage | Dna polymerase | Dna repair | Dna replication | Dna-binding | Dna-directed dna polymerase | Dpo4 | Magnesium | Metal-binding | Mutator protein | Nucleotidyltransferase | O6-methylguanine | Transferase | Transferase/dna complex | Translesion dna synthesis
