Replication of phase fd RF with fd gene 2 protein and phage T4 enzymes.

T.F Meyer,I Bäumel,K Geider,P Bedinger

doi:10.1016/s0021-9258(19)69279-3

Abstract

Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes. As anticipated from previous in vitro studies carried out with this system (Morris, C. F., Sinha, N. K., and Alberts, B. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism. We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made. The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system. The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme. This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA.

Highlights

Bacteriophage fd replicative form DNA with a nick tendency of the T7 DNA polymerase stwoitch strands, which in the viral strandserves as a template forDNA repli- diminished the yield of single-stranded DNA drastically (6)
A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism.We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made
The gene 2 protein reduces thelevel of DNA synthesis cleaved by gene 2 protein provides an appropriate substrate by2-foldat low concentrations, even though it only for this T4 DNA replication system since fthdeenzyme does cleaves the DNA products efficiently at higherlevels of not remain attached to thecleaved DNA (4,5 ),in contrast to the enzyme

Summary

RESULTS

In part by the payment of page charges. This article must be herebymarked “aduertisement” in accordance with 18 U.S.C. 0. Box 100, nicked by the fd gene 2 protein. Analysis of these reaction products by electron microscopy shows that replication occursin FIG.[2]. Electron microscopic pictures of typical replication a rolling circle mode, beginning by covalent addition of nucleotides onto the 3’OH end of the nick, with concomitant parental strand displacementas the polymerasemoves in the products. A, a rolling circle with a single-stranded tailof about unit length besidesa linear molecule of unit length. Without active gen2eprotein present during taken with the electron microscope Philips EM 400 at the magnifithe DNA synthesis, the rollingcircle products have single- cation of: X 8350.

Analysis of the replication products made in the presence

No of molecules counted

DISCUSSION