Differential dynamics of ATR-mediated checkpoint regulators

The ATR-Chk1 checkpoint pathway is activated by UV-induced DNA lesions and replication stress. Little was known about the spatio and temporal behaviour of the proteins involved, and we, therefore, examined the behaviour of the ATRIP-ATR and Rad9-Rad1-Hus1 putative DNA damage sensor complexes and the downstream effector kinase Chk1. We developed assays for the generation and validation of stable cell lines expressing GFP-fusion proteins. Photobleaching experiments in living cells expressing these fusions indicated that after UV-induced DNA damage, ATRIP associates more transiently with damaged chromatin than members of the Rad9-Rad1-Hus1 complex. Interestingly, ATRIP directly associated with locally induced UV damage, whereas Rad9 bound in a cooperative manner, which can be explained by the Rad17-dependent loading of Rad9 onto damaged chromatin. Although Chk1 dissociates from the chromatin upon UV damage, no change in the mobility of GFP-Chk1 was observed, supporting the notion that Chk1 is a highly dynamic protein.