Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention.
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.
Lukas
Claudia
C
Danish Cancer Society, Institute of Cancer Biology, Copenhagen, Denmark. lukas@biobase.dk
Melander
Fredrik
F
Stucki
Manuel
M
Falck
Jacob
J
Bekker-Jensen
Simon
S
Goldberg
Michal
M
Lerenthal
Yaniv
Y
Jackson
Stephen P
SP
Bartek
Jiri
J
Lukas
Jiri
J
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006834:Hydrazines; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D012516:Osteosarcoma / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C417561:Alexa 488 hydrazide; D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C468783:H2AX protein, human; D006657:Histones; D006834:Hydrazines; C413163:MDC1 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D015534:Trans-Activators