Vidi, P.-A. | bioRxiv Science

bioRxiv2017

NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

Accumulation of 53BP1 at DNA breaks determines DNA repair pathway choice and promotes checkpoint activation. Here, we show regulation of 53BP1 beyond repair foci. 53BP1 movements are constrained in the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma, NuMA prevents 53BP1 accumulation at DNA breaks and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a new mechanism that involves the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

cell biology↗

bioRxiv2017

Structured illumination to spatially map chromatin motions

We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in the nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340{+/-}30 nm, which simultaneously photoactivate a 7x7 matrix pattern of GFP-labeled histones, with spots 1.70 m apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals DNA damage-dependent decrease in chromatin diffusion. The DOE instrumentation can easily and cheaply be implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in non-adherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

cell biology↗