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

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

doi:10.1016/j.celrep.2021.110176

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

Cell Rep. 2021 Dec 28;37(13):110176. doi: 10.1016/j.celrep.2021.110176.

Authors

Barbara Martinez-Pastor¹, Giorgia G Silveira², Thomas L Clarke², Dudley Chung³, Yuchao Gu⁴, Claudia Cosentino⁵, Lance S Davidow⁶, Gadea Mata⁷, Sylvana Hassanieh⁵, Jayme Salsman³, Alberto Ciccia⁸, Narkhyun Bae⁹, Mark T Bedford¹⁰, Diego Megias⁷, Lee L Rubin⁶, Alejo Efeyan¹¹, Graham Dellaire¹², Raul Mostoslavsky¹³

Affiliations

¹ The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; Molecular Oncology Program, Spanish National Cancer Research Center (CNIO), Madrid 28029, Spain. Electronic address: bmartinezp@cnio.es.
² The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
³ Department of Pathology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
⁴ School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
⁵ The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA.
⁶ Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
⁷ Confocal Microscopy Unit, Spanish National Cancer Research Center (CNIO), Madrid 28029, Spain.
⁸ Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA; Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA.
⁹ Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA.
¹⁰ Department of Epigenetics & Molecular Carcinogenesis, M.D.Anderson Cancer Center, University of Texas, Smithville, TX 78957, USA.
¹¹ Molecular Oncology Program, Spanish National Cancer Research Center (CNIO), Madrid 28029, Spain.
¹² Department of Pathology, Dalhousie University, Halifax, NS B3H 4R2, Canada; Beatrice Hunter Cancer Research Institute, Halifax, NS B3H 4R2, Canada. Electronic address: dellaire@dal.ca.
¹³ The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address: rmostoslavsky@mgh.harvard.edu.

Abstract

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.

Keywords: DNA damage foci; DNA repair; GOF; PHF20; chromatin accessibility; high throughput; laser microirradiation.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Chromatin / genetics*
Chromatin / metabolism
DNA Damage*
DNA Repair Enzymes / genetics
DNA Repair Enzymes / metabolism*
DNA Repair*
High-Throughput Screening Assays
Histones / genetics
Histones / metabolism*
Humans
Kinetics
Open Reading Frames*
Tumor Suppressor p53-Binding Protein 1 / genetics
Tumor Suppressor p53-Binding Protein 1 / metabolism*

Substances

Chromatin
Histones
TP53BP1 protein, human
Tumor Suppressor p53-Binding Protein 1
DNA Repair Enzymes

Abstract

Publication types

MeSH terms

Substances

Grants and funding