Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein.
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.
Bernstein
J A
JA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Richardson
C C
CC
D015153:Blotting, Western; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D019915:DNA Primase; D017886:Durapatite; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D006882:Hydroxyapatites; D008970:Molecular Weight; D009843:Oligoribonucleotides / Q000138:chemical synthesis; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology
D029968:Escherichia coli Proteins; D006882:Hydroxyapatites; D009843:Oligoribonucleotides; D014764:Viral Proteins; D017886:Durapatite; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C027093:TraI protein, E coli; D004265:DNA Helicases