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Journal of Nucleic Acids
Volume 2010, Article ID 319142, 16 pages
Research Article

Differential Dynamics of ATR-Mediated Checkpoint Regulators

1Department of Cell Biology and Genetics, Cancer Genome Center, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
2Department of Radiation Oncology, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
3Unidad de Investigación, Hospital Universitario de Canarias, Ofra s/n, La Laguna 38320, Tenerife, Spain

Received 3 May 2010; Accepted 28 June 2010

Academic Editor: Ashis Basu

Copyright © 2010 Daniël O. Warmerdam et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.