Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal.pdf

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Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal.pdf

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Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal

Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal Carcinoma Cells in Response to Replication-dependent DNA Double Strand Breaks* Received for publication, April 19, 2006, and in revised form, August 10, 2006 Published, JBC Papers in Press, August 10, 2006, DOI 10.1074/jbc.M603747200 Haruyuki Takemura1, V. Ashutosh Rao1, Olivier Sordet1, Takahisa Furuta, Ze-Hong Miao, LingHua Meng, Hongliang Zhang, and Yves Pommier2 From the Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-4255 The Mre11Rad50Nbs1 (MRN) complex binds DNA dou- ble strand breaks to repair DNA and activate checkpoints.We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to campto- thecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomer- ase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independ- ent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphoryl- ation of Mre11 and Nbs1. Mre11 complementation in MRN- deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Pro- teasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 lev- els. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediate