Acute reduction of an origin recognition complex (ORC) subunit in human cells reveals a requirement of ORC for Cdk2 activation.
The origin recognition complex (ORC) is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G1 phase. At the G1/S transition, elevated cyclin E-CDK2 activity triggers 1DNA replication to enter S phase. The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK. However, how the CDK cycle is coordinated with replication initiation remains elusive. Here we report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity. This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G1. p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition. Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis. However, in some cells the release of the CDK2 block caused catastrophic events like apoptosis. Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication. These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability.
Machida
Yuichi J
YJ
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Teer
Jamie K
JK
Dutta
Anindya
A
D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D002478:Cells, Cultured; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D047468:Immunoprecipitation; D008938:Mitosis; D008954:Models, Biological; D051738:Origin Recognition Complex; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016196:S Phase; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D001973:Bromodeoxyuridine