Focus in numbers: Characterizing protein oligomerization dynamics at DNA double strand breaks.

Abstract

During radiotherapy, ionizing radiation (IR) is used to kill cancer cells by directly inducing high levels of genotoxic DNA double-strand breaks (DSB). These breaks are quickly detected and bound by DNA damage sensor proteins that activate a cascade of events leading to the recruitment and accumulation of repair factors (e.g. 53BP1) at sites of damage in dot-like structures called foci. The balance and interplay between the plethora of proteins recruited at the break site determine the choice of the repairing pathway, which can have paramount consequences on cell fate and ultimately in the response to cancer radiotherapy. Indeed, substantial evidence indicates that enhanced DSB repair (DSBR) capacity in individual patients is a major determinant in tumour radioresistance, while both radiosensitivity and the occurrence of radiotherapy-induced secondary cancers, are presumably associated with reduced DSBR function. Therefore, there is a pressing need to better understand the fundamental mechanisms of DSBR, towards improving the clinical management of patients undergoing radiotherapy. While it is clear that DNA repair relies on the balanced contribution of various repair factors, the oligomerization dynamics governing their organization into foci remain largely unknown. Here we combine Spatial Intensity Distribution Analysis (SpIDA) with an automated high-content foci detection algorithm to quantify the number of 53BP1 molecules at DSBR foci in cells challenged with increasing doses of the radiomimetic drug zeocin. Consistently with published data, 53BP1 foci number increases linearly at lower zeocin doses while plateauing at higher DNA damage load (zeocin concentration above 500 μg/mL). At this plateau, despite the availability of a nucleoplasmic protein pool, the number of 53BP1 molecules per focus decreases, suggesting a further not yet clarified layer of dynamic regulation potentially leverageable as therapeutical target.