Rate-Limiting Steps in the DNA Polymerase I Reaction Pathway

S. J. Benkovic, V. Mizrahi, R. N. Henrie, J. F. Marlier, K. A. Johnson

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Abstract

The initial rates of incorporation of dTTP and thymidine 5'-O-(3-thiotriphosphate) (dTTPaS) into poly(dA)·oligo(dT) during template-directed synthesis by the large fragment of DNA polymerase I have been measured by using a rapid-quench technique. The rates were initially equal, indicating a non-rate-limiting chemical step. However, the rate of thionucleotide incorporation steadily diminished to 10% of its initial value as the number of consecutive dTMPaS residues in the primer strand increased. This anomalous behavior can be attributed to the helix instability inherent in phosphorothioate-containing duplexes. Positional isotope exchange experiments employing the labeled substrate [α-18O2]dATP have revealed negligible α,β-bridging →β-nonbridging isotope exchange in template-directed reactions of Escherichia coli DNA polymerase I (Pol I) both in the presence and in the absence of added inorganic pyrophosphate (PPi), suggesting rapid PP; release following the chemical step. These observations are consistent with a rate-limiting step that is tentatively assigned to a conformational change of the E-DNA-dNTP complex immediately preceding the chemical step. In addition, the substrate analogue (Sp)-dATPαS has been employed to examine the mechanism of the PPj exchange reaction catalyzed by Pol I. The net retention of configuration at the α-P is interpreted in terms of two consecutive inversion reactions, namely, 3'-hydroxyl attack, followed by PP; attack on the newly formed primer terminus. Kinetic analysis has revealed that while α-phosphorothioate substitution has no effect upon the initial rate of polymerization, it does attenuate the PPi exchange reaction by a factor of 15-18-fold. The mechanistic implications of these results are discussed.

Original languageEnglish (US)
Pages (from-to)4010-4018
Number of pages9
JournalBiochemistry
Volume24
Issue number15
DOIs
Publication statusPublished - Jul 1 1985

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All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Benkovic, S. J., Mizrahi, V., Henrie, R. N., Marlier, J. F., & Johnson, K. A. (1985). Rate-Limiting Steps in the DNA Polymerase I Reaction Pathway. Biochemistry, 24(15), 4010-4018. https://doi.org/10.1021/bi00336a031