TY - JOUR
T1 - DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells
AU - Jeyasekharan, Anand D.
AU - Ayoub, Nabieh
AU - Mahen, Robert
AU - Ries, Jonas
AU - Esposito, Alessandro
AU - Rajendra, Eeson
AU - Hattori, Hiroyoshi
AU - Kulkarni, Rajan P.
AU - Venkitaraman, Ashok R.
PY - 2010/12/14
Y1 - 2010/12/14
N2 - 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.
AB - 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.
KW - DNA damage response
KW - Fluorescence spectroscopy
KW - Protein dynamics
KW - Single-molecule imaging
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U2 - 10.1073/pnas.1009577107
DO - 10.1073/pnas.1009577107
M3 - Article
C2 - 21098284
AN - SCOPUS:78650749457
SN - 0027-8424
VL - 107
SP - 21937
EP - 21942
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 50
ER -