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 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.
Kadyrov
F A
FA
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA. kadyrov@niehs.nih.gov
Drake
J W
JW
D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D018741:Replication Origin / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D015678:Viral Structural Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase