Size classes of products synthesized processively by two subassemblies of Escherichia coli DNA polymerase III holoenzyme.

P J Fay,K O Johanson,C S Mchenry,R A Bambara

doi:10.1016/s0021-9258(19)83833-4

Abstract

Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form. These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase III holoenzyme. In a previous publication (Fay, P. J., Johanson K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT)10 . poly(dA) template, the DNA polymerase III holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Now we show that DNA polymerase III' adds 30 to 40 nucleotides before dissociation. This number can be increased to approximately 60 if spermidine is present, but it is insensitive to the presence of E. coli single-stranded DNA-binding protein. DNA polymerase III adds about 50 nucleotides before dissociation, but this value can be increased to 200 nucleotides in the presence of the binding protein. Using measurement of product sizes made on an oligo(dT)10 . poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III and the beta subunit was monitored. Finally, it is shown that the products obtained from a purified initiation complex of holoenzyme and oligo(dT)10 . poly(dA) derive solely from the holoenzyme.

Highlights

Two forms of Escherichia coli DNA polymerase 111, DNA polymerase 111’, and DNA polymerase III* have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form
Chem 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT),o. poly(dA)template, the DNA polymeraseI11 holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides
The above results demonstrate that each stable form of DNA polymerase I11 makes a distinct class of product sizes in the absence or presence of effectors of synthesis, which is Effects of Spermidine and SSB on Synthetic Activity of indicative of the particular enzyme form

Summary

Introduction

Two forms of Escherichia coli DNA polymerase 111, DNA polymerase 111’, and DNA polymerase III* have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form These forms of DNA polymerase I11 are intermediate in complexity between the core DNA polymerase 111 and the DNA polymerase I11 holoenzyme. Poly(dA)template, the DNA polymeraseI11 holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Using measurement of product sizes made on an oligo(dT)lo-poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III* and the p subunit was monitored. The DNA polymerase 111holoenzyme of Escherichia coli is a complex, multisubunit enzyme composed of a DNA polymerase 111core and several auxiliary proteins. To catalyzelimited synthesison nuclease-activatedduplex DNA, is not active by itself on the naturally primed phage DNA templates

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Conclusion