Control of DNA Replication and Chromosome Ploidy by Geminin and Cyclin A

Abstract

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.

Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2

“…This fits well with a crucial role of cyclin B/CDK1 complexes in this phenotype, probably together with cyclin A/CDK1 complexes. Indeed, re-replication has been reported after cyclin A ablation in drosophila, and also after cyclin A knock down together with Emi-1 in mammals (Mihaylov et al, 2002;Machida and Dutta, 2007). As cyclin A and B/ CDK1 complexes are activated in a temporal succession, the data provided here, together with these two articles, lead to the idea that cyclin A would participate in early re-replication inhibition in mammals controlled by CDK1, whereas cyclin B complexes would inhibit re-replication later.…”

Geminin Deficiency Causes a Chk1-dependent G2 Arrest inXenopus

“…There may be differences between the two organisms in the way checkpoint controls are exercised. It has recently been reported that cultured Drosophila cells accumulate excessive amounts of DNA (up to 8n) when geminin synthesis is inhibited by interfering RNA (Mihaylov et al, 2002). This may indicate that Drosophila cells are more dependent on a geminin-requiring mechanism to inhibit rereplication than vertebrate cells.…”

RNA polymerase II transcription is required for human papillomavirus type 16 E7- and hydroxyurea-induced centriole overduplication

“…These control experiments show that the transfection of cells with siRNA duplexes targeting cyclin A2 at 24 h before exposure to HU does not lead to a cell cycle arrest. Lastly, depletion of cyclin A2 has been suggested to cause changes in ploidy (Mihaylov et al, 2002). However, there is no indication so far that cells with altered ploidy cannot undergo additional rounds of centriole duplication (Mantel et al, 1999;Guarguaglini et al, 2005).…”