A divalent FHA/BRCT-binding mechanism couples the MRE11-RAD50-NBS1 complex to damaged chromatin.
The MRE11-RAD50-NBS1 (MRN) complex accumulates at sites of DNA double-strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1-dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation.
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich, Switzerland.
Flurina J
FJ
Hari
Christoph
C
Spycher
Stephanie
S
Jungmichel
Lucijana
L
Pavic
Manuel
M
Stucki
D017766:Acid Anhydride Hydrolases; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016195:G2 Phase; D006801:Humans; D000076228:MRE11 Homologue Protein; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C413163:MDC1 protein, human; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D015534:Trans-Activators; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes