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

Escherichia coli DNA polymerase III core enzyme and DNA polymerase I11 holoenzyme synthesize DNA via a processive mechanism, but size classes of products made are very different for the two enzyme forms and are influenced by template structure and other conditions.We have employed three methods to measure the size of newly synthesized DNA made after one association of the DNA polymerase with DNA primer termini.The first, a biochemical kinetic approach (Bambara, R., Uyemura, D., and Choi, T. (1978) J. Biol.Chem 253, 413-423), was used with a randomly primed fd DNA template.Results are obtained as the average size of newly synthesized products.The core enzyme adds approximately 11 nucleotides to the primers before dissociation.This value can be reduced severalfold by spermidine.The holoenzyme adds at least 100 nucleotides before dissociation.This number is increased in the presence of spermidine or single-stranded DNAbinding protein.In the second method, gel filtration is used to estimate the size of products made in the presence of excess poly(dA)=oligo(dT), such that various classes of product sizes can be detected.The core enzyme produces a single size class 10 to 15 nucleotides long, but shorter products when spermidine is present.Preparations of DNA polymerase 111 holoenzyme produce two apparent size classes, one 10 to 30 nucleotides in length which is attributable to free endogenous core DNA polymerase I11 and the other more than 100 nucleotides in length.The sizes and proportions of these classes can be influenced by spermidine, single-stranded DNA-binding protein, and antibody to the / 3 subunit of the holoenzyme.The third method involves detection of the product made from dnaG primase-primed G4 DNA in the presence of single-stranded DNA-binding protein.Product size is monitored by the appearance of Hue 111 restriction endonuclease sites.The holoenzyme can be shown to synthesize a full length product on 6 4 DNA (5577 nucleotides) after a single binding event.Escherichia coli contains three DNA polymerases, desig-GM-24441 to R.