Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures.
An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.
Tatsumi
Y
Y
Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Tsurimoto
T
T
Shirahige
K
K
Yoshikawa
H
H
Obuse
C
C
D000911:Antibodies, Monoclonal / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D003850:Deoxyribonuclease I / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D017830:Octoxynol / Q000494:pharmacology; D051738:Origin Recognition Complex; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D003902:Detergents; C497223:ORC1 protein, human; D051738:Origin Recognition Complex; D012333:RNA, Messenger; D012965:Sodium Chloride; D017830:Octoxynol; D004247:DNA; D003851:Deoxyribonucleases; D003850:Deoxyribonuclease I