Cyclin A2/Cdk1 is Essential for the in vivo S Phase Entry by Phosphorylating Top2a

Abstract

1 Cyclin-dependent kinase 1 (CDK1) plays essential roles in cell cycle regulation. 2 However, due to the early embryonic lethality of mouse Cdk1 mutants, the in vivo 3 role of CDK1 in regulating cell cycle and embryonic development remains unclear. 4Here, by generating zebrafish cdk1 mutants using CRISPR/Cas9 system, we show 5 that cdk1 -/embryos exhibit severe microphthalmia accompanied with multiple 6 defects in polarized cell division, S phase entry and M phase progression, cell 7 apoptosis and cell differentiation, but not in interkinetic nuclear migration (IKNM). 8By informatics analysis, we identified Top2a as a potential downstream target, and 9Cyclin A2 and Cyclin B1 as partners of Cdk1 in cell cycle. Depletion of either 10 Cyclin A2 or Top2a leads to decreased S phase entry and increased DNA damage 11 response in zebrafish retinal cells, and depletion of Cyclin B1 leads to M phase 12 arrest. Immunoprecipitation shows that Cdk1 and Cyclin A2 physically interact in 13 vivo. Moreover, phosphorylation of Top2a on Serine 1213 (S1213) site is almost 14 absent in either cdk1 or ccna2 mutants, but in not ccnb1 mutants. Furthermore, 15 overexpression of TOP2A S1213 , the phosphomimetic form of human TOP2A, rescues 16 S phase entry and microphthalmia defects in cdk1 -/and ccna2 -/embryos. Taken 17 together, our data suggests that Cdk1 interacts with Cyclin A2 to regulate S phase 18 entry through phosphorylating Top2a, and with Cyclin B1 to regulate M phase 19 progression in vivo. 20