In vivo dynamics of BRCA2 and Rad51. (360G-Wellcome-080346_Z_06_Z)

WT Studentship 4-year PhD Project Grant Project title: In vivo dynamics of BRCA2 and Rad51 A combination of biochemical, structural, cell biological and biophysical data have suggested a model whereby BRCA2 is able to sequester Rad51 monomers, with direct binding holding them at highly conserved motifs within exon 11 (the BRC repeats: Bignell et all 1997; Wong et al., 1997). However in response to DNA damage, BRCA2 is thought to directly facilitate Rad51 targeting and oligomerisation during HR. The biophysical support for this model comes from a fluorescence recovery after photobleaching (FRAP) study of GFP tagged Rad51. Rad51 was detected in an immobile fraction in living cells due to self association and also due to the interaction with BRCA2, and it was the BRCA2 population which was found to specifically increase in mobility in response to DNA damage. Biochemical data showing that the expression of individual BRCA2 BRC repeats can block Rad51 filament formation to varying degrees in vitro is also in agreement with this model. However, it raises a further question about the significance of this observed heterogeneity in the Rad51 binding regions, which is also implicit in the observation that mutations in different BRC repeats are associated with varying risks of carcinogenesis. To study the dynamic switch in BRCA2 function from sequestration to mobilization, recent unpublished work in the Venkitaraman lab has begun to study the in vivo dynamics of the protein using FCS. FCS measures fluctuations in fluorescence intensity inside an illuminated volume in the order of one femtoliter, using a confocal microscopy setup. Through a time correlation of the signal, the hydrodynamic and photophysical properties of a fluorescently tagged species can be determined as it diffuses through the observation volume. Typically, mobility coefficients and local concentrations are calculated. Although less well established than other non invasive fluorescence-based imaging techniques such as FRAP, FCS provides more powerful insight into certain parameters.