The origin recognition complex marks a replication origin in the human TOP1 gene promoter.
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.
Keller
Christian
C
Department of Biology, University of Konstanz, 78464 Konstanz, Germany. Christian.Keller@uni-konstanz.de
Ladenburger
Eva-Maria
EM
Kremer
Marcel
M
Knippers
Rolf
R
D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D004276:DNA, Satellite / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015226:Dinucleoside Phosphates; D006801:Humans; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018741:Replication Origin
D002843:Chromatin; D003432:Cross-Linking Reagents; D004264:DNA Topoisomerases, Type I; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D051738:Origin Recognition Complex; C015772:CpG dinucleotide