DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells

Anand D. Jeyasekharan, Nabieh Ayoub, Robert Mahen, Jonas Ries, Alessandro Esposito, Eeson Rajendra, Hiroyoshi Hattori, Rajan P. Kulkarni, Ashok R. Venkitaraman

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)21937-21942
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number50
DOIs
StatePublished - Dec 14 2010
Externally publishedYes

Keywords

  • DNA damage response
  • Fluorescence spectroscopy
  • Protein dynamics
  • Single-molecule imaging

ASJC Scopus subject areas

  • General

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