Three-dimensional imaging reveals the spatial separation of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes at ionizing radiation-induced foci.
OBJECTIVE
Ionizing radiation (IR)-induced DNA damage causes the accumulation of DNA damage response (DDR) proteins as visible foci in cell nuclei. Despite the identified functional roles in DNA repair, the spatial relationships of different DDR proteins at foci have not been explicitly examined. This study aims to systematically compare the distribution of DDR proteins at IR-induced foci.


METHODS
MCF-7 cells were treated with IR, stained for γH2AX, MDC1, RNF8, RNF168, 53BP1, Abraxas (CCDC98), BRCA1, BRCC36, Merit40 (NBA1) and RAP80, and then imaged using high-resolution three-dimensional (3-D) confocal microscopy to assess the relative localization of proteins at foci.


RESULTS
All BRCA1-A complex components displayed strong co-localization, which overlapped significantly with RNF8 and RNF168, but not with γH2AX and MDC1. Intriguingly, 53BP1 co-located well with γH2AX and MDC1, but remained separate from RNF8 and RNF168. These co-localization patterns were consistent for at least 3h after IR.


CONCLUSIONS
The foci formations of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes are spatially independent. Such divergence was not anticipated from prior studies on the recruitment of these proteins to foci. This information indicates that individual foci may represent distinct sites of DNA repair facilitated by a specific subset of DDR proteins.
Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia. mythmok@mythmok.com
Myth T S
MT
Mok
Beric R
BR
Henderson
D048868:Adaptor Proteins, Signal Transducing; D001943:Breast Neoplasms / Q000378:metabolism*; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004249:DNA Damage; D059766:DNA End-Joining Repair; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism* / Q000528:radiation effects; D009687:Nuclear Proteins / Q000378:metabolism* / Q000528:radiation effects; D011839:Radiation, Ionizing; D059767:Recombinational DNA Repair; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D000071857:Tumor Suppressor p53-Binding Protein 1; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C486521:RNF8 protein, human; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; C111164:BRAP protein, human; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases