TY - JOUR
T1 - A lysine residue in the fingers subdomain of T7 DNA polymerase modulates the miscoding potential of 8-Oxo-7,8-dihydroguanosine
AU - Brieba, Luis G.
AU - Kokoska, Robert J.
AU - Bebenek, Katarzyna
AU - Kunkel, Thomas A.
AU - Ellenberger, Tom
N1 - Funding Information:
We thank Annie Heroux and the staff of beam line X26-C for technical assistance, and Ying Li, John Pascal, Oleg Tsodikov, Andre L.B. Ambrosio, and Hector Viadiu for their advice. This work was supported by a grant from the National Institute of Health (GM55390) to T.E. and by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health. T.E. is the Hsien Wu and Daisy Yen Wu Professor of Biological Chemistry and Molecular Pharmacology. L.G.B. is indebted to the Pew Latin American Postdoctoral program for its generous support.
PY - 2005/11
Y1 - 2005/11
N2 - 8-Oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG(syn)·dA(anti) Hoogsteen base pairs. DNA polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG(syn)·dATP mismatch in the polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.
AB - 8-Oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG(syn)·dA(anti) Hoogsteen base pairs. DNA polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG(syn)·dATP mismatch in the polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.
UR - http://www.scopus.com/inward/record.url?scp=27644597370&partnerID=8YFLogxK
U2 - 10.1016/j.str.2005.07.020
DO - 10.1016/j.str.2005.07.020
M3 - Article
C2 - 16271888
AN - SCOPUS:27644597370
SN - 0969-2126
VL - 13
SP - 1653
EP - 1659
JO - Structure
JF - Structure
IS - 11
ER -