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J. Biol. Chem., Vol. 277, Issue 12, 10354-10361, March 22, 2002
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From the Origin recognition complex (ORC), CDC6, and
MCM proteins assemble sequentially to form prereplication
chromatin. However, their organization remains largely unclear in
mammalian cells. Here we show that ORC1 proteins are associated with
non-chromatin nuclear structures and assemble in nuclear foci in
mammalian cells using an in vivo chemical cross-linking
method. CDC6 proteins were also found to assemble in nuclear foci on
non-chromatin nuclear structures, although their physical association
with ORC1 has been undetectable. In contrast to the situation in yeast
cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder
binds to nuclease-sensitive chromatin. Further analyses indicate that
ORC2 physically interacts with ORC1 on non-chromatin nuclear
structures. On the other hand, our results suggest that although a
small proportion of MCM complexes are loaded onto chromatin regions
near ORC foci, most of them are more widely distributed. Possible
relations between such organization of prereplication chromatin and
complicated origin specification in higher eukaryotic cells are discussed.
Nuclear Organization of DNA Replication Initiation Proteins in
Mammalian Cells*
§,,, and
Laboratory of Viral Oncology, Division of
Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden,
Chikusa-ku, Nagoya 464-8681 and the ¶ Mitsubishi Kagaku Institute
of Life Sciences, 11 Minamiooya, Machida,
Tokyo 194-8511, Japan
*
This work was supported in part by grants from the Ministry
of Education, Science, Sports, and Culture of Japan and from the Uehara
Memorial Foundation.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.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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