Rereplication by depletion of geminin is seen regardless of p53 status and activates a G2/M checkpoint.
Genomic DNA replication is tightly controlled to ensure that DNA replication occurs once per cell cycle; loss of this control leads to genomic instability. Geminin, a DNA replication inhibitor, plays an important role in regulation of DNA replication. To investigate the role of human geminin in the maintenance of genomic stability, we eliminated geminin by RNA interference in human cancer cells. Depletion of geminin led to overreplication and the formation of giant nuclei in cells that had wild-type or mutant p53. We found that overreplication caused by depletion of geminin activated both Chk1 and Chk2, which then phosphorylated Cdc25C on Ser216, resulting in its sequestration outside the nucleus, thus inhibiting cyclin B-Cdc2 activity. This activated G(2)/M checkpoint prevented cells with overreplicated DNA from entering mitosis. Addition of caffeine, UCN-01, or inhibitors of checkpoint pathways or silencing of Chk1 suppressed the accumulation of overreplicated cells and promoted apoptosis. From these results, we conclude that geminin is required for suppressing overreplication in human cells and that a G(2)/M checkpoint restricts the proliferation of cells with overreplicated DNA.
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Wenge
W
Zhu
Yuefeng
Y
Chen
Anindya
A
Dutta
D017209:Apoptosis / Q000502:physiology; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002110:Caffeine / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D019926:Cyclin B / Q000378:metabolism; D004261:DNA Replication; D004789:Enzyme Activation; D064248:Geminin; D018816:Genes, cdc; D042822:Genomic Instability; D006657:Histones / Q000378:metabolism; D006801:Humans; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000378:metabolism
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; C577995:GMNN protein, human; D064248:Geminin; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D002110:Caffeine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases