Nuclear organization of DNA replication initiation proteins in mammalian cells.
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.
Fujita
Masatoshi
M
Laboratory of Viral Oncology, Division of Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. mfujita@aichi-cc.jp
Ishimi
Yukio
Y
Nakamura
Hiromu
H
Kiyono
Tohru
T
Tsurumi
Tatsuya
T
D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D051738:Origin Recognition Complex; D011233:Precipitin Tests; D016196:S Phase; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D003902:Detergents; D009687:Nuclear Proteins; C497223:ORC1 protein, human; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D017830:Octoxynol; D004247:DNA; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7