Structure of a second BRCT domain identified in the nijmegen breakage syndrome protein Nbs1 and its function in an MDC1-dependent localization of Nbs1 to DNA damage sites.
The Nijmegen breakage syndrome protein Nbs1 is a component of the MRN (Mre11-Rad50-Nbs1) complex, central to the DNA damage response. While Nbs1 is generally believed to encompass a forkhead-associated domain linked to a breast cancer C-terminal (BRCT) domain, to date there is no experimental information on its three-dimensional structure. Through nuclear magnetic resonance (NMR) three-dimensional structure determination, we demonstrate that there is a second BRCT domain (BRCT2) in Nbs1. The domain has the characteristic BRCT topology, but with a long insertion shown to be flexible by NMR relaxation measurements. In the absence of sequence similarity to other proteins, a search for structural analogs of BRCT2 returned the second BRCT domain of the tandem BRCT repeats of cell cycle checkpoint proteins MDC1 (mediator of DNA damage checkpoint protein 1) and BRCA1 (breast cancer protein 1), suggesting that like MDC1 and BRCA1, Nbs1 also possesses tandem BRCT domains with phosphoprotein binding ability. Structure-based single point mutations in human Nbs1 were evaluated in vivo and revealed that BRCT2 is essential for an MDC1-dependent relocalization of Nbs1 to DNA damage sites, most likely through a direct interaction of Nbs1 tandem BRCT domains with phosphorylated MDC1.
Xu
Chao
C
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Wu
Liming
L
Cui
Gaofeng
G
Botuyan
Maria Victoria
MV
Chen
Junjie
J
Mer
Georges
G
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004249:DNA Damage / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020080:Tandem Repeat Sequences / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D002247:Carbon Isotopes; D018797:Cell Cycle Proteins; C413163:MDC1 protein, human; C511600:NBN protein, Xenopus; C112156:NBN protein, human; D009587:Nitrogen Isotopes; D009687:Nuclear Proteins; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins