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J. Biol. Chem., Vol. 276, Issue 31, 29559-29566, August 3, 2001
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and
From the Laboratory of Molecular Genetics, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709-2233
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.
To whom correspondence should be addressed: Laboratory of
Molecular Genetics E3-01, NIEHS, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-3029; Fax: 919-541-7613; E-mail: kadyrov@niehs.nih.gov.
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