Characterization of the Helicase and Primase Activities of the 63-kDa Component of the Bacteriophage T7 Gene 4 Protein

J A Bernstein,C C Richardson

doi:10.1016/s0021-9258(18)51596-9

Abstract

Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein. Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass. We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 396-400). The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells. The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein. The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein. The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase. Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold. This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein.

Highlights

The preparation contains 5-10% contaminating 56kDa protein, as shown by Western analysis using polyclonal antibodies to thepurified 56-kDa protein
The preferential recovery of the 56-kDa protein most likely reflects a combination of proteolysis of the 63-kDa polypeptide during isolation, and a bias introduced by pooling of chromatographic fractions based upon activity in the DNA synthesis assays used to purify the 56-kDa/63-kDa gene 4 proteins
The 63-kDa protein has helicase activity and synthesizessequence-specific oligonucleotides. It interacts with T7 DNA polymerase as indicated by its ability to facilitate DNA synthesis a t a preformed replication fork as well as by its capacity to stimulate the elongation of tetraribonucleotide primers by T7 DNA polymerase

Summary

THEJOURNALOF BIOLOGICACHLEMISTRY

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No 22, Issue of August 5, pp. 1306613073,1989 Printed in U.S.A. The preferential recovery of the 56-kDa protein most likely reflects a combination of proteolysis of the 63-kDa polypeptide during isolation, and a bias introduced by pooling of chromatographic fractions based upon activity in the DNA synthesis assays used to purify the 56-kDa/63-kDa gene 4 proteins. Such a bias is expected in view of the ability of an excess of the 56-.

Total primase Specific primase activityactivity w

Trimers Tetromers

DNA synthesis”

DISCUSSION

Findings

EXPERIMENTAL PROCEDURES