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J. Biol. Chem., Vol. 277, Issue 47, 44886-44897, November 22, 2002
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From the Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720
Large T-antigen (T-ag) is a viral helicase
required for the initiation and elongation of simian virus 40 DNA
replication. The unwinding activity of the helicase is powered by ATP
hydrolysis and is critically dependent on the oligomeric state of the
protein. We confirmed that the double hexamer is the active form of the helicase on synthetic replication forks. In contrast, the single hexamer cannot unwind synthetic forks and remains bound to the DNA as
ATP is hydrolyzed. This inability of the T-ag single hexamer to release
the DNA fork is the likely explanation for its poor helicase activity.
We characterized the interactions of T-ag single and double hexamers
with synthetic forks and single-stranded (ss) DNA. We demonstrated that
DNA forks promote the formation of T-ag double hexamer. The lengths of
the duplex region and the 3' tail of the synthetic forks are the
critical factors in assembly of the double hexamer, which is bound to a
single fork. We found that the cooperativity of T-ag binding to ss
oligonucleotides increased with DNA length, suggesting that multiple
consecutive subunits in the hexamer engage the ssDNA.
Characterization of Simian Virus 40 T-antigen Double Hexamers
Bound to a Replication Fork
THE ACTIVE FORM OF THE HELICASE*
,
*
This work was supported in part by National Institutes of
Health Grant GM31655 (to N. R. C.) and CA42414 (to M. R. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Fellow of the Program in Mathematics and Molecular Biology and
supported by a Burroughs Welcome Fund fellowship.
§
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, Division of Biochemistry and Molecular Biology, 401 Barker Hall 3204, Berkeley, CA 94720-3204. Tel.: 510-642-5266; Fax:
510-643-1079; E-mail: ncozzare@socrates.berkeley.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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