BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells |
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| Authors: | Marcel Reuter Alex Zelensky Ihor Smal Erik Meijering Wiggert A van Cappellen H Martijn de Gruiter Gijsbert J van Belle Martin E van Royen Adriaan B Houtsmuller Jeroen Essers Roland Kanaar Claire Wyman |
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| Institution: | 1Department of Genetics, Cancer Genomics Centre Netherlands, 2Department of Medical Informatics, 3Department of Radiology, 4Erasmus Optical Imaging Centre, 5Department of Pathology, 6Department of Vascular Surgery, and 7Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands |
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| Abstract: | Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2. |
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