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Volume 270,
Number 15,
Issue of April 14, pp. 8910-8919, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differences
in the Mechanism of Stimulation of T7 DNA Polymerase by Two Binding
Modes of Escherichia coli Single-stranded DNA-binding Protein
Mary N.
Rigler
,
Louis J.
Romano
Escherichia coli single-stranded DNA-binding protein
( Eco SSB) has been shown previously to display several DNA
binding modes depending on the ionic conditions. To determine what
effect these various binding modes have on DNA replication, we have
studied DNA synthesis by the T7 DNA polymerase under ionic conditions
where Eco SSB interacts with either 72 or 91 nucleotides of
M13 DNA. These forms presumably correspond to the previously described
(SSB) and (SSB) (Lohman and Ferrari, 1994)
that were determined using the binding of SSB to homopolymers. Here we
report the stimulation induced by (SSB) to be 4-fold
greater than that produced by (SSB) under conditions where
the template is in large excess. Surprisingly, when the polymerase
level is raised so that it is in molecular excess, (SSB) no longer stimulates synthesis while (SSB) affords a
4-fold stimulation, which is the same level of stimulation as when the
template was in excess. Both SSB forms increase the rate of DNA
synthesis and were found to stimulate synthesis by relieving template
secondary structures. However, (SSB) specifically
increases strand displacement synthesis, while (SSB) stimulates synthesis by increasing the affinity of the polymerase
for the template.

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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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