Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint

Abstract

The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase–polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling. We find that CDC45, CLSPN and MCM7 are novel CK2α interacting partners and these interactions are particularly important for maintenance of stable MCM7–CDC45, ATRIP–ATR–MCM7, and ATR–CLSPN protein complexes. Consistently, cells depleted of CK2α and treated with hydroxyurea display compromised replisome integrity, reduced chromatin binding of checkpoint mediator CLSPN, attenuated ATR-mediated S-phase checkpoint and delayed recovery of stalled forks. In further support of this, differential gene expression analysis by RNA-sequencing revealed that down-regulation of CK2α accompanies global shutdown of genes that are implicated in the S-phase checkpoint. These findings add to our understanding of the molecular mechanisms involved in DNA replication by showing that the protein kinase CK2α is essential for maintaining the stability of the replisome machinery and for optimizing ATR-CHK1 signalling activation upon replication stress.

Phosphorylation inhibits intramolecular interactions, DNA-binding and protein interactions of Claspin through disordered/ structured conformation transition

“…We reported that Cdc7, in conjunction with CK1λ1, can phosphorylate CKBD, and that Cdc7 play a predominant role in cancer cells, whereas CK1λ1 plays a major role in normal cells 19 . Furthermore, casein kinase (CK2) interacts with the replisome containing Claspin and may regulate the checkpoint activation 31 . To understand how phosphorylation affects the structure and functions of Claspin, the effects of various kinases including Cdc7, CK1λ1, Chk1, and CK2 were examined.…”

“…Since Claspin is very rich in IDRs which are preferred targets of phosphorylation, it can be phosphorylated at multiple sites. Indeed, Claspin can be phosphorylated by Cdc7, CK1λ1, ATR, and Chk1, and also by other kinases, such as Polo-like kinase 1, stress-activated protein kinase (SAPK) p38 and CK2α 3,31 . In this report, we have shown that Claspin is directly phosphorylated by Cdc7, Ck1λ1, Chk1, and CK2α kinases in vitro ( Fig.…”

“…We reported that Cdc7, in conjunction with CK1g1, can phosphorylate CKBD, and that Cdc7 play a predominant role in cancer cells, whereas CK1g1 plays a major role in normal cells 19 . Furthermore, casein kinase (CK2) interacts with the replisome containing Claspin and may regulate the checkpoint activation 31 .…”

“…Among them, CK2 is implicated in the phosphorylation of repair proteins, which are platform proteins for the recruitment of other proteins to the damaged DNA for repair 38 . It was reported that CK2a interacts with Claspin and CMG, and perturbation of the CK2a level results in defective S-phase checkpoint activation 31 .…”

Phosphorylation inhibits intramolecular interactions, DNA-binding and protein interactions of Claspin through disordered/ structured conformation transition

“…Since Claspin is very rich in IDRs which are preferred targets of phosphorylation, it can be phosphorylated at multiple sites. Indeed, Claspin can be phosphorylated by Cdc7, CK1g1, ATR, and Chk1, and also by other kinases, such as Polo-like kinase 1, stress-activated protein kinase (SAPK) p38 and CK2a 3,31 . In this report, we have shown that Claspin is directly phosphorylated by Cdc7, Ck1g1, Chk1, and CK2a kinases in vitro (Fig.…”