title: DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells.
abstract: How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.
authors.affiliation: The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 0XZ, United Kingdom.
authors.forename: Anand D
authors.initials: AD
authors.lastname: Jeyasekharan
authors.forename: Nabieh
authors.initials: N
authors.lastname: Ayoub
authors.forename: Robert
authors.initials: R
authors.lastname: Mahen
authors.forename: Jonas
authors.initials: J
authors.lastname: Ries
authors.forename: Alessandro
authors.initials: A
authors.lastname: Esposito
authors.forename: Eeson
authors.initials: E
authors.lastname: Rajendra
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Hattori
authors.forename: Rajan P
authors.initials: RP
authors.lastname: Kulkarni
authors.forename: Ashok R
authors.initials: AR
authors.lastname: Venkitaraman
mesh_terms: D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002645:Chickens; D004249:DNA Damage; D018390:Gene Targeting; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
chemical_list: D024682:BRCA2 Protein; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051135:Rad51 Recombinase