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 language | English (US) |
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Pages (from-to) | 4010-4018 |
Number of pages | 9 |
Journal | Biochemistry |
Volume | 24 |
Issue number | 15 |
DOIs | |
State | Published - Jul 1 1985 |
All Science Journal Classification (ASJC) codes
- Biochemistry