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J. Biol. Chem., Vol. 277, Issue 13, 11174-11183, March 29, 2002
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From the We previously identified and purified from human
(HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding
specificity for cruciform DNA regardless of its sequence. DNA
cruciforms have been implicated in the regulation of initiation of DNA
replication. CBP is a member of the 14-3-3 family of proteins, which
are conserved regulatory molecules expressed in all eukaryotes. Here,
the in vivo association of CBP/14-3-3 with mammalian
origins of DNA replication was analyzed by studying its association
with the monkey replication origins ors8 and
ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3
The Human Cruciform-binding Protein, CBP, Is Involved in DNA
Replication and Associates in Vivo with Mammalian
Replication Origins*
§,§,, and§
McGill Cancer Center and
§ Department of Biochemistry, McGill University, Montreal,
Quebec H3G 1Y6, Canada and the ¶ Department of Genetics, the
Hospital for Sick Children, University of Toronto,
Toronto, Ontario M5G 1X8, Canada
, -
, -
, and -
isoforms with these origins was found to be
~9-fold higher, compared with other portions of the genome, in
logarithmically growing cells. In addition, the association of these
isoforms with ors8 and ors12 was also analyzed
as a function of the cell cycle. Higher binding of 14-3-3, -,
-, and - isoforms with ors8 and ors12 was
found at the G1/S border, by comparison with other stages
of the cell cycle. The CBP/14-3-3 cruciform binding activity was also
found to be maximal at the G1/S boundary. The involvement
of 14-3-3 in mammalian DNA replication was analyzed by studying the
effect of anti-14-3-3, -, -, and - antibodies in the
in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3, -,
and - antibodies alone or in combination inhibited p186 replication
by ~50-80%, while anti-14-3-3 antibodies had a lesser effect
(~25-50%). All of the antibodies tested were also able to interfere
with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA
replication by binding to cruciform-containing molecules, and
dissociates after origin firing.
*
This work was supported by grants from the Canadian
Institutes of Health Research (to M. Z.-H.) and the Cancer Research
Society (to M. Z.-H. and G. B. P.).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.
To whom correspondence should be addressed: McGill Cancer
Center, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-3537; Fax: 514-398-6769; E-mail: mzannis@med.mcgill.ca.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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