Conditional Coupling of Leading-strand and Lagging-strand DNA Synthesis at Bacteriophage T4 Replication Forks

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided t...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2001-08, Vol.276 (31), p.29559-29566
Main Authors: Kadyrov, F A, Drake, J W
Format: Article
Language:English
Subjects:
Bacteriophage T4 - enzymology
Bacteriophage T4 - genetics
Base Sequence
DNA Replication
DNA, Viral - biosynthesis
DNA, Viral - genetics
DNA-Binding Proteins - metabolism
DNA-Directed DNA Polymerase - metabolism
Escherichia coli - genetics
Escherichia coli - virology
Genes, Viral
Kinetics
Molecular Sequence Data
Oligodeoxyribonucleotides - chemistry
Oligodeoxyribonucleotides - metabolism
Phage T4
Plasmids
Replication Origin - genetics
Viral Proteins - genetics
Viral Proteins - metabolism
Viral Structural Proteins - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M101310200