Cell cycle-dependent regulation of the association between origin recognition proteins and somatic cell chromatin.
Previous studies have suggested that cell cycle-dependent changes in the affinity of the origin recognition complex (ORC) for chromatin are involved in regulating initiation of DNA replication. To test this hypothesis, chromatin lacking functional ORCs was isolated from metaphase hamster cells and incubated in Xenopus egg extracts to initiate DNA replication. Intriguingly, Xenopus ORC rapidly bound to hamster somatic chromatin in a Cdc6-dependent manner and was then released, concomitant with initiation of DNA replication. Once pre-replication complexes (pre-RCs) were assembled either in vitro or in vivo, further binding of XlORC was inhibited. Neither binding nor release of XlORC was affected by inhibitors of either cyclin-dependent protein kinase activity or DNA synthesis. In contrast, inhibition of pre-RC assembly, either by addition of Xenopus geminin or by depletion of XlMcm proteins, augmented ORC binding by inhibiting ORC release. These results demonstrate a programmed release of XlORC from somatic cell chromatin as it enters S phase, consistent with the proposed role for ORC in preventing re-initiation of DNA replication during S phase.
Sun
Wei-Hsin
WH
National Institute of Child Health and Human Development, Building 6/416, National Institutes of Health, Bethesda, MD 20892-2753, USA.
Coleman
Thomas R
TR
DePamphilis
Melvin L
ML
D000818:Animals; D042846:CDC2-CDC28 Kinases; D016466:CHO Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D006224:Cricetinae; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008297:Male; D051738:Origin Recognition Complex; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014981:Xenopus; D029867:Xenopus Proteins
C118388:Cdc6 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases