Enzymological characterization of KB cell DNA polymerase-alpha. Regulation of template binding by nucleic acid base composition.

P.A Fisher,J.T Chen,D Korn

doi:10.1016/s0021-9258(19)70108-2

Abstract

Enzymological characterization of KB cell DNA polymerase-alpha. Regulation of template binding by nucleic acid base composition.

Highlights

Ase-a for single-stranded DNA templates is dependent on the base composition of those templates
The data presented in this paper indicate thatthe intrinsic affinity of polymerase-a for single-stranded polydeoxynucleotides is dependent on base composition
The kinetic studies have all been performed with polymerase concentrations of the order of 0.1 n~ to satisfy the requirement of the Briggs-Haldane steady state assumption that substrate concentration ([SI) be much greater than total enzyme concentration ([Eltow).Rate determinations were based on 5-min incubations under reaction conditions in which polymerase activity was linear with time for at least 20 min; the rates arceonsidered to be validexpressionsof initial velocity

Summary

EXPERIMENTAL PROCEDURES

The sources of most of the materials were as previously noted [35,7]. Poly(dG), 7.4 S,d(pT),, d(pA)r, and d(pC)w, ere purchased from Collaborative Research. All other homopolymers and synthetic heteropolymerswere prepared as described under “Methods”. [3H]dTTP was from New England Nuclear; [3H]dATP, [3H]dCTP, [’HIdGTP, [a-”PJdTTP, and[a-32P]dATPwere from Amersham/Searle. Spleen phosphodiesterase 11, pancreatic DNase I, and micrococcal nuclease were from Worthington and were used without further purification. Single strand-specific nucleases SI and Neurospora crassa endonuclease were obtained from Bethesda Research Laboratories and Miles Laboratories, respectively, and were used as supplied. Phage $,X 174 single-stranded circular DNA was prepared according to the method of Franke and Ray [8]and was found by alkaline agarose gel electrophoresis to be >90% intact circles. Terminal deoxynucleotidyltransferase, purified from calfthymus, was the gift of Dr R.

Methods

RESULTS

Findings

DISCUSSION