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Papers In Press, published online ahead of print May 9, 2002
Biologie, Universität Konstanz, Konstanz 76464
Corresponding Author: christian.keller{at}uni-konstanz.de
The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analysed 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 characterised matrix attachment regions (MAR). The MAR elements analysed 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 (TOM 1) and an alphoid satellite sequence which underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that ORC on this site may perform functions other than replication initiation.
J. Biol. Chem, 10.1074/jbc.M202165200
Submitted on March 5, 2002
Revised on April 10, 2002
Accepted on May 9, 2002
The origin recognition complex marks a replication origin in the human TOP1 gene promoter
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