Control of DNA replication and chromosome ploidy by geminin and cyclin A.
Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, we used the RNA silencing-interference technique (RNAi) to eliminate the Drosophila geminin homologue from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. We found that the silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. We observed that the silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, our studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells.
Mihaylov
Ivailo S
IS
Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Kondo
Takeshi
T
Jones
Lynn
L
Ryzhikov
Sophia
S
Tanaka
Junko
J
Zheng
Jianyu
J
Higa
Leigh Ann
LA
Minamino
Naoto
N
Cooley
Lynn
L
Zhang
Hui
H
D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D002875:Chromosomes / Q000187:drug effects / Q000378:metabolism*; D019925:Cyclin A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019926:Cyclin B / Q000037:antagonists & inhibitors / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation; D004330:Drosophila; D029721:Drosophila Proteins; D005434:Flow Cytometry; D020868:Gene Silencing / Q000187:drug effects; D008969:Molecular Sequence Data; D011003:Ploidies; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D018797:Cell Cycle Proteins; C503698:CycB protein, Drosophila; D019925:Cyclin A; D019926:Cyclin B; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded; D004247:DNA; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1