DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics.
We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.
Potapova
Olga
O
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Chan
Chikio
C
DeLucia
Angela M
AM
Helquist
Sandra A
SA
Kool
Eric T
ET
Grindley
Nigel D F
ND
Joyce
Catherine M
CM
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D020029:Base Pairing; D002384:Catalysis; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism
D004256:DNA Polymerase I; D000225:Adenine; D013941:Thymine