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J. Biol. Chem., Vol. 276, Issue 9, 6337-6342, March 2, 2001
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From the We investigated the association of human origin
recognition complex (ORC) proteins hOrc1p and hOrc2p with
chromatin in HeLa cells. Independent procedures including limited
nuclease digestion and differential salt extraction of isolated nuclei
showed that a complex containing hOrc1p and hOrc2p occurs in a
nuclease-resistant compartment of chromatin and can be eluted with
moderate high salt concentrations. A second fraction of hOrc2p that
dissociates in vitro at low salt conditions was found to
occur in a chromatin compartment characterized by its high
accessibility to micrococcal nuclease. Functional differences between
these two sites become apparent in HeLa cells that synchronously enter
the S phase after a release from a double-thymidine block. The
hOrc1p/hOrc2p-containing complexes dissociate from their chromatin
sites during S phase and reassociate at the end of mitosis. In
contrast, the fraction of hOrc2p in nuclease-accessible, more open
chromatin remains bound during all phases of the cell cycle. We propose
that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell
cycle-dependent release of the hOrc1p/hOrc2p-containing
complexes is in line with previous studies with Xenopus and
Drosophila systems, which indicated that a change in
ORC stability occurs after prereplication complex formation.
This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.
The Human Origin Recognition Complex Protein 1 Dissociates from
Chromatin during S Phase in HeLa Cells*
§,¶,, and
Department of Biology, Universität
Konstanz, D-78457 Konstanz and the ¶ GSF-Haematologikum,
Marchioninistrasse 25 D-81377 München, Germany
*
This work was supported by the Deutsche
Forschungsgemeinschaft.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.
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