The primase activity of DNA polymerase alpha from calf thymus.

F Grosse,G Krauss

doi:10.1016/s0021-9258(18)89675-2

Abstract

The nearly homogeneous 9 S DNA polymerase alpha from calf thymus contains a primase activity that allows priming of DNA synthesis on single-stranded templates in the presence of ribonucleoside triphosphates. Both on synthetic and natural single-stranded templates, RNA primers of 8-15 nucleotides in length are formed. In the absence of dNTPs, primers of some hundred nucleotides in length are observable. ATP and/or GTP are required for the priming reaction. UTP and CTP cannot initiate the RNA synthesis. M13 single-stranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Priming occurs mostly at specific sites on the M13 genome and replication products of up to 6000 nucleotides in length are formed. In the presence of the single-stranded DNA binding protein from Escherichia coli, specificity of priming is strongly increased. The primase is inhibited by salt and actinomycin; it is insensitive to alpha-amanitin and N-ethylmaleimide. Due to the strong complex formation between DNA polymerase and primase, it has not been possible to separate the two activities of the multisubunit 9 S enzyme.

Highlights

The nearly homogeneous9 S DNA polymerasea from 14)
We have studied the template utilization, the lengthof the RNA primer, and thsepecificity of theprimasereaction usinghomopolymers and singlestranded M13 DNA asa template
One unit of debate during the lasyt ears. This is mainly due to the sensi- primase activity isdefined as the amountof enzyme that leads to an tivity of DNA polymerase a to endogenous proteolysis

Summary

Introduction

Part of the ambiguitieshave been resolved and it isnow calf thymus contains a primase activity that allows commonly accepted that DNApolymerase a contains a large priming of DNAsynthesis on single-stranded templates subunit of 140-180 kDa associated with smaller subunits of in the presence of ribonucleoside triphosphates. Both 40-70 kDa. We have purified a 9 S DNA polymerase a from on synthetic and natural single-stranded templates, calf thymus to nearhomogeneity which contains subunitsof. M13 singlestranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Erase and primase, it has not been possible to separate mmol) were from New England Nuclear.Oligonucleotides and hothe two activitiesof the multisubunit S enzyme

Methods

Results

Conclusion