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J. Biol. Chem., Vol. 277, Issue 35, 31430-31440, August 30, 2002
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,
From the Department of Biology, University of Konstanz,
78464 Konstanz, Germany
The locations of the origin recognition complex
(ORC) in mammalian genomes have been elusive. We have therefore
analyzed the DNA sequences associated with human ORC via in
vivo cross-linking and chromatin immunoprecipitation. Antibodies
specific for hOrc2 protein precipitate chromatin fragments that also
contain other ORC proteins, suggesting that the proteins form
multisubunit complexes on chromatin in vivo. A binding
region for ORC was identified at the CpG island upstream of the human
TOP1 gene. Nascent strand abundance assays show that the
ORC binding region coincides with an origin of bidirectional
replication. The TOP1 gene includes two well characterized
matrix attachment regions. The matrix attachment region elements
analyzed contain no ORC and constitute no sites for replication
initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC
sites in the human genome, we isolated a sequence close to another
actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this
site may perform functions other than replication initiation.
To whom correspondence should be addressed. Tel.:
49-7531-884238; Fax: 49-7531-884036; E-mail:
Christian.Keller@uni-konstanz.de.
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