The asterisks denote a statistically significant difference between the G2/M populations comparing WT to T355A transfected cells (p=0.0092). HeLa cells depleted for TLK1 also display prolonged G2/M checkpoint activation in response to IR. that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, Pitolisant oxalate and that cells lacking TLK1 display a prolonged G2/M arrest upon Pitolisant oxalate exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response. == Introduction == Cell cycle checkpoints comprise an elaborate network of signal transduction pathways that maintain the proper order of cellular events [1]. Checkpoints can initiate a delay in progression through the cell cycle in response to both endogenous and exogenous DNA damage, thus granting the cell time to repair damage and ensuring that damaged DNA is not replicated and passed on to subsequent generations [2,3]. The importance of proper checkpoint function is underscored by the fact that mutations in checkpoint genes can lead to genetic instability and are found in a host of human cancers and cancer predisposition syndromes [48]. The human 9-1-1 complex is a proliferating SPTAN1 cell nuclear antigen (PCNA)- like heterotrimeric DNA clamp composed of Rad9, Rad1, and Hus1 [911]. The 9-1-1 complex is loaded onto DNA at 5-recessed ends, a common substrate resulting Pitolisant oxalate from DNA metabolism and damage [1214]. Chromatin-bound 9-1-1 is thought to act as a scaffold that localizes other elements of the checkpoint machinery to sites of DNA damage and thus plays a critical role in initiating and maintaining the checkpoint response [15,16]. In particular, the 9-1-1 complex recruits DNA topoisomerase 2-binding protein 1 (TopBP1) to damage-induced lesions, thus facilitating activation of the PI3K-related kinase ataxia telangiactasia and Rad3-related (ATR) [1719]. The 9-1-1 TopBP1 ATR module is required for efficient activation of Chk1 [20], a checkpoint kinase that inhibits Cdc25 phosphatase activity and delays the transition from G2 to mitosis [2123]. Rad9 is unique among the other components of the 9-1-1 complex in that it possesses an unstructured C-terminal tail of approximately 110 amino acids that does not share homology with Rad1, Hus1 or PCNA [2427]. This region is not required for 9-1-1 complex formation [11,28] but it is necessary for TopBP1 association and proper checkpoint function [15,17,29]. In addition, it is heavily modified by phosphorylation both constitutively and transiently in response to cell cycle position and DNA damage, and hence represents a potential regulatory mechanism for checkpoint control [15,3032]. For example, S272 is phosphorylated rapidly and transiently in response to damage regardless of cell cycle position by the PI3K-related kinase ataxia telangiactasia-mutated (ATM) [15,33], T292 is targeted during mitosis by Cdc2 [30], and S341 and S387 are both phosphorylated constitutively and are required for Pitolisant oxalate the interaction between Rad9 and TopBP1 [15,31]. While our lab and others have made progress towards identifying the targeted residues within Rad9 and the context under which they are modified, our understanding is far from complete. The complex and interdependent nature of Rad9 phosphorylation has made the task of establishing the physiological significance of these events challenging. The human Tousled-like kinases 1 & 2 (TLK1 and TLK2) are homologues ofArabidopsis thalianaTousled, and they exhibit peak activity in S-phase and likely participate in chromatin remodeling [34,35]. TLK1 and TLK2 are thought to oligermerize [35], Pitolisant oxalate and both phosphorylate the H3/H4 histone chaperone anti-silencing function 1 homolog A (ASF1A), which itself facilitates histone deposition and chromatin assembly during S-phase and following DNA repair[3639]. The physiological significance of TLK-dependent phosphorylation of ASF1A is poorly understood, although there is evidence that it may serve to protect it from proteasomal degradation [40]. TLK1 in particular is inactivated rapidly in response to double-stranded.