Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G(1) transition in mammals.
To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G(1) phase, and reappeared as cells progressed through G(1) phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G(1) phase, but became stably bound during mid-G(1) phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed.
Natale
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DA
National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD 20892-2753, USA.
Li
C J
CJ
Sun
W H
WH
DePamphilis
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ML
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D008322:Mammals / Q000235:genetics* / Q000378:metabolism; D008677:Metaphase; D051379:Mice; D064126:Minichromosome Maintenance Complex Component 3; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009865:Oocytes / Q000737:chemistry; D051738:Origin Recognition Complex; D018741:Replication Origin; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014020:Tissue Extracts / Q000494:pharmacology; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C066470:MCM3 protein, human; C513269:Mcm3 protein, mouse; D009687:Nuclear Proteins; C497223:ORC1 protein, human; C497224:ORC2 protein, human; C497228:Orc1 protein, mouse; D051738:Origin Recognition Complex; D014020:Tissue Extracts; D064126:Minichromosome Maintenance Complex Component 3