Human CDK2 inhibition modifies the dynamics of chromatin-bound minichromosome maintenance complex and replication protein A.
Minichromosome maintenance (MCM) proteins form a complex and possess helicase activity to unwind the DNA duplex and establish a replication fork. To assure that origins only fire once per cell cycle, the MCM complex is removed from chromatin and inactivated as cells exit S phase. In this report, we demonstrate that CDK2 depletion in human cells leads to an overall phosphorylation defect at mitosis with increased rereplication, correlated with the accumulation of chromatin-bound MCM proteins. We show that CDK2 suppression results in decreased MCM4 phosphorylation at multiple serine and threonine sites. In addition, CDK2 inhibition induces an increase in chromatin-bound replication protein A (RPA) which should bind to single-stranded DNA regions, possibly establishing a replication intermediate that activates the ATR cascade. Finally, we observe that loss of CDK2 function in G1 delays replication initiation while it promotes rereplication in G2/M. Thus, by modulating the phospho-status of MCM4 and regulating origin firing, S phase CDK2 appears to be an integrated component of cellular machinery required for temporally controlling replication activity and maintaining genomic stability.
Zhu
Yonghong
Y
Department of Discovery Research, DNAX Research, Incorporated, Palo Alto, California 94304, USA. yonghongzhu@dnax.org
Ishimi
Yukio
Y
Tanudji
Marcel
M
Lees
Emma
E
D015153:Blotting, Western; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors*; D004247:DNA / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D016193:G1 Phase; D016195:G2 Phase; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008297:Male; D026341:Minichromosome Maintenance 1 Protein / Q000737:chemistry* / Q000378:metabolism; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry*; D012190:Retroviridae / Q000235:genetics; D016196:S Phase; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D002843:Chromatin; D004791:Enzyme Inhibitors; D026341:Minichromosome Maintenance 1 Protein; D034741:RNA, Small Interfering; D051716:Replication Protein A; D013912:Threonine; D012694:Serine; D004247:DNA; D051357:Cyclin-Dependent Kinase 2; D015739:Nocodazole