Volume 270,
Number 22,
Issue of June 2, pp. 13384-13391, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly
of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp
Loader, and Sliding Clamps in One Holoenzyme Particle
V. FOUR DIFFERENT POLYMERASE-CLAMP COMPLEXES ON DNA
P. Todd
Stukenberg
,
Mike
O'Donnell
Several different subassemblies of DNA polymerase III holoenzyme
can be purified from Escherichia coli. Toward the goal of
understanding the functional significance of these subassemblies, we
have used the 
complex clamp loader and the 
ring to assemble
each different polymerase onto DNA. Through use of radioactive labeled
proteins, the subunit structure of each resulting processive polymerase
has been determined. Use of DNA polymerase III core, the complex,
and results in a core- complex on DNA; the complex is
not incorporated into the structure. The addition of 
to the
assembly reaction to form either core
-
or
core- results in a more efficient
polymerase and more stabile association of core- on DNA,
although the complex still does not remain on DNA. The
complex clamp loader was retained on DNA with the other subunits only
if it was first assembled into the polymerase (Pol) III* structure. The
clamp loader within Pol III* appeared to be capable of loading two
clamps onto DNA for both core polymerases within Pol III*,
consistent with the hypothesis that one replicase can simultaneously
replicate both strands of a duplex chromosome. These findings extend
those of an earlier study showing that distinctive polymerases can be
assembled depending on the presence or absence of (Maki, S., and
Kornberg, A.(1988) J. Biol. Chem. 263, 6561-6569). The
significance of these distinct polymerases in separate paths of DNA
metabolism is discussed.
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