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(Received for publication, June 11, 1997)
From the Department of Biochemistry, School of Hygiene and Public
Health, Johns Hopkins University, Baltimore, Maryland 21205
We have used a set of bacteriophage
Volume 272, Number 45,
Issue of November 7, 1997
pp. 28800-28813
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Properties of Replication Fork Assemblies Established
by the Bacteriophage
O and P Replication Proteins
and
Escherichia coli replication proteins to establish rolling
circle DNA replication in vitro to permit characterization
of the functional properties of replication forks. We demonstrate
that the replication fork assembly synthesizes leading strand DNA
chains at a physiological rate of 650-750 nucleotides/s at 30 °C.
This rate is identical to the fork movement rate we obtained using a
minimal protein system, composed solely of E. coli DnaB
helicase and DNA polymerase III holoenzyme. Our data are consistent
with the conclusion that these two key bacterial replication proteins
constitute the basic functional unit of a replication fork. A
comparison of rolling circle DNA replication in the minimal and replication systems indicated that DNA synthesis proceeded for more
extensive periods in the system and produced longer DNA chains,
which averaged nearly 200 kilobases in length. The higher potency of
the replication system is believed to result from its capacity to
mediate efficient reloading of DnaB helicase onto rolling circle
replication products, thereby permitting reinitiation of DNA chain
elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually
stimulated rolling circle DNA replication, but they apparently act
indirectly by blocking accumulation of inhibitory free single-stranded
DNA product. Finally, in the course of this work, we discovered that
E. coli DNA polymerase III holoenzyme is itself capable of
carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. coli
single-stranded DNA-binding protein.
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