387회 Cell cycle arrest upon defect of initiation of human chromosome r…

387회

연사 :  황 동 수, 박 종 훈, Department of Biological Sciences and the Institute of Molecular Biology and Genetics

제목:  Cell cycle arrest upon defect of initiation of human chromosome replication

Abstract

  Through M to G1 phase of mammalian cell cycle, chromosome replication initiatiation starts by assembly of the proteins ORC, Cdc6, Cdt1 and MCM2-7 onto replication origins. The resulting complex is pre-replication complex (pre-RC). At G1/S boundary, MCM10 and TopBP1 functions are considered to be necessary to allow Cdc45 to be loaded onto the pre-RC complex to assemble pre-initiation complex (pre-IC). In S phase, DNA polymerases a, d and e are loaded on the pre-IC and duplicate chromosomes.  To understand the interrelation of chromosome initiation process with cell cycle, we inhibited the formation of pre-IC by depleting MCM10 or TopBP1 by using siRNA methodand determined the fate of cell cycle.

MCM10-depleted cells exhibited slow progression of S phase followed by G2 arrest.  The depleted cells exhibited a defect in DNA replication from mid and late S phase origins. No increase of phospho-H3 indicated that the treated cells were not able to enter M phase. The G2 arrest appearsto be caused by incomplete replication of chromosome, which resulted in the activation of Chk1 and Chk2 followed by the inhibition of CDK1. These results suggest that the replication initiation at the mid and the late S origins requires MCM10 function to complete chromosome replication.

In contrast to the G2 arrest of MCM10 depletion, depletion of TopBP1 arrested cells at G1 phase with low cyclin E/CDK2 kinase activity, even though the level of cyclin E protein increased. The low cyclin E/CDK2 kinase activity resulted from the up-regulation of CDK inhibitors, p21 and p27, in a p53-independent manner.  Also, Cdc7/Dbf4 kinase activity, which is indispensable for the conversion of pre-RC to pre-IC, was low in the TopBP1 depleted cells.  Knock-down of both p21 and p27 in the TopBP1-depleted cells restored both cyclinE/CDK2 and Cdc7/Dbf4 kinase activities, although the depletion could not allow the cells to progress into S phase. These results suggest that TopBP1 regulates p21 and p27 for the G1/S progression in addition to its role in the formation pre-IC.