Summary information and primary citation
- PDB-id
-
2j6t;
DSSR-derived features in text and
JSON formats
- Class
- transferase-DNA
- Method
- X-ray (2.6 Å)
- Summary
- Ternary complex of sulfolobus solfataricus dpo4 DNA
polymerase, o6- methylguanine modified DNA, and datp.
- Reference
-
Eoff RL, Irimia A, Egli M, Guengerich FP (2007):
"Sulfolobus
Solfataricus DNA Polymerase Dpo4 is Partially Inhibited
by "Wobble" Pairing between O6- Methylguanine and
Cytosine, But Accurate Bypass is Preferred."
J.Biol.Chem., 282, 1456. doi:
10.1074/JBC.M609661200.
- Abstract
- 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.
