PMID:16411765
| Citation |
Potapova, O, Chan, C, DeLucia, AM, Helquist, SA, Kool, ET, Grindley, ND and Joyce, CM (2006) DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics. Biochemistry 45:890-8 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2567902 Online version:10.1021/bi051792i |
| Keywords |
Adenine/analogs & derivatives; Adenine/metabolism; Base Pairing; Catalysis; DNA Polymerase I/chemistry; DNA Polymerase I/genetics; DNA Polymerase I/metabolism; DNA Replication; Hydrogen Bonding; Kinetics; Models, Molecular; Thymine/analogs & derivatives; Thymine/metabolism |
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