Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention

• DOI: 10.1038/sj.emboj.7600269
• Publication Date: 2004-06-17T09:01:24Z
• Authors: Claudia Lukas; Fredrik Melander; Manuel Stucki; Jacob Falck; Simon Bekker-Jensen; Michal Goldberg; Yaniv Lerenthal; Stephen P Jackson; Jiri Bartek; Jiri Lukas
• Keywords: Cell Nucleus; Osteosarcoma; 0303 health sciences; Microscopy, Confocal; Antibodies, Monoclonal; Fluorescent Antibody Technique; Nuclear Proteins; Cell Cycle Proteins; Phosphoproteins; Chromatin; Protein Structure, Tertiary; DNA-Binding Proteins; Histones; 03 medical and health sciences; Hydrazines; Cell Line, Tumor; Humans; RNA Interference; RNA, Small Interfering; Adaptor Proteins, Signal Transducing; DNA Damage; Fluorescent Dyes

Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11-Rad50-Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double-strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1-independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1's FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB-flanking chromatin and caused aberrant pan-nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB-induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA-mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB-flanking chromatin. Our data suggest that Mdc1 functions as an H2AX-dependent interaction platform enabling a switch from transient, Mdc1-independent recruitment of Nbs1 to DSBs towards sustained, Mdc1-dependent interactions with the surrounding chromosomal microenvironment.