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Papers In Press, published online ahead of print June 4, 2001
J. Biol. Chem, 10.1074/jbc.M101310200
Submitted on February 12, 2001
Revised on June 1, 2001
Accepted on June 4, 2001
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709
Corresponding Author: kadyrov{at}niehs.nih.gov
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/synthesis 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.
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