Abstract
A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization.
Highlights
How the dynamic three-dimensional organization of the mammalian nucleus influences fundamental processes like DNA repair is unclear [1,2]
We have studied in this context the role of A-type lamins, the intermediate-filament proteins lamin A and C encoded by LMNA, which form a structural scaffold at the nuclear membrane and within the nuclear interior [3,4] thought to bind to chromatin [5]
Each fusion protein was detected in western blots as a single species with the expected molecular mass using several different antibodies (Fig. 1A). dsRed-53BP1TD formed visible foci in unchallenged cells as previously reported [19], which increased in number after the treatment of cells with ionising radiation (IR), and were confirmed as sites of DNA damage by their co-localization with staining for the serine-139 phosphorylated form of histone H2AX
Summary
How the dynamic three-dimensional organization of the mammalian nucleus influences fundamental processes like DNA repair is unclear [1,2]. DsRed-53BP1TD accumulated in foci at sites of DNA damage induced within the nuclei of living cells either with a 405 nm laser after pre-sensitization with Hoechst 33342, or with a pulsed 365 nm micropoint laser.
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