Nucleic Acids Research, Vol 26, Issue 24 5636-5643, Copyright © 1998 by Oxford University Press
C Iftode and JA Borowiec
The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to
induce the melting of 8 bp within the SV40 origin of replication. We found
previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central
8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured
by human replication protein A (hRPA). To understand the mechanism by which
hRPA denatures these pseudo-origin molecules, as well as the role that hRPA
plays during the initiation of SV40 DNA replication, we characterized the
key parameters for the pseudo-origin binding and denaturation reactions.
The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x
10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length
ssDNA under the same reaction conditions. The binding and denaturation of
PO- 8 occurred with different kinetics with the rate of binding determined
to be approximately 4-fold greater than the rate of denaturation. Although
hRPA binding to PO-8 was relatively temperature independent, an increase in
incubation temperature from 4 to 37 degreesC stimulated denaturation nearly
4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those
substrate molecules bound by hRPA, showing that hRPA can bind the
pseudo-origin substrate without causing its complete denaturation. Tests of
other single-stranded DNA-binding proteins (SSBs) over a range of SSB
concentrations revealed that the ability of the SSBs to bind the
pseudo-origin substrate, rather than denature the substrate, correlated
best with the known ability of these SSBs to support the T
antigen-dependent SV40 origin-unwinding activity. Our data indicate that
hRPA first binds the DNA substrate using a combination of contacts with the
ssDNA bubble and duplex DNA flanks and then, on only a fraction of the
bound substrate molecules, denatures the DNA substrate.
ARTICLES
Unwinding of origin-specific structures by human replication protein A occurs in a two-step process
Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA.
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