title: BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells.
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.
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Marcel
authors.initials: M
authors.lastname: Reuter
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Zelensky
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Ihor
authors.initials: I
authors.lastname: Smal
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Meijering
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Wiggert A
authors.initials: WA
authors.lastname: van Cappellen
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: H Martijn
authors.initials: HM
authors.lastname: de Gruiter
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Gijsbert J
authors.initials: GJ
authors.lastname: van Belle
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Martin E
authors.initials: ME
authors.lastname: van Royen
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Essers
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands.
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands Department of Genetics, Cancer Genomics Centre Netherlands, Department of Medical Informatics, Department of Radiology, Erasmus Optical Imaging Centre, Department of Pathology, Department of Vascular Surgery, and Department of Radiation Oncology, Erasmus University Medical Centre, 3000 CA Rotterdam, Netherlands c.wyman@erasmusmc.nl.
authors.forename: Claire
authors.initials: C
authors.lastname: Wyman
mesh_terms: D000818:Animals; D024682:BRCA2 Protein / Q000378:metabolism*; D004249:DNA Damage; D053595:Embryonic Stem Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D057507:Mice, 129 Strain; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D066329:Protein Aggregates; D011485:Protein Binding; D021381:Protein Transport; D051135:Rad51 Recombinase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D059010:Single-Cell Analysis
chemical_list: D024682:BRCA2 Protein; C106711:BRCA2 protein, mouse; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse