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Assessing kinetics and recruitment of DNA repair factors using high content screens.

Cell reports (2021-12-30)
Barbara Martinez-Pastor, Giorgia G Silveira, Thomas L Clarke, Dudley Chung, Yuchao Gu, Claudia Cosentino, Lance S Davidow, Gadea Mata, Sylvana Hassanieh, Jayme Salsman, Alberto Ciccia, Narkhyun Bae, Mark T Bedford, Diego Megias, Lee L Rubin, Alejo Efeyan, Graham Dellaire, Raul Mostoslavsky

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

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