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

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

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

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.

Original language	English (US)
Article number	110176
Journal	Cell Reports
Volume	37
Issue number	13
DOIs	https://doi.org/10.1016/j.celrep.2021.110176
State	Published - Dec 28 2021
Externally published	Yes

Keywords

DNA damage foci
DNA repair
GOF
PHF20
chromatin accessibility
high throughput
laser microirradiation

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

MD Anderson CCSG core facilities

Protein Array and Analysis Core

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10.1016/j.celrep.2021.110176

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Martinez-Pastor, B., Silveira, G. G., Clarke, T. L., Chung, D., Gu, Y., Cosentino, C., Davidow, L. S., Mata, G., Hassanieh, S., Salsman, J., Ciccia, A., Bae, N., Bedford, M. T., Megias, D., Rubin, L. L., Efeyan, A., Dellaire, G., & Mostoslavsky, R. (2021). Assessing kinetics and recruitment of DNA repair factors using high content screens. Cell Reports, 37(13), Article 110176. https://doi.org/10.1016/j.celrep.2021.110176

Martinez-Pastor, B, Silveira, GG, Clarke, TL, Chung, D, Gu, Y, Cosentino, C, Davidow, LS, Mata, G, Hassanieh, S, Salsman, J, Ciccia, A, Bae, N, Bedford, MT, Megias, D, Rubin, LL, Efeyan, A, Dellaire, G & Mostoslavsky, R 2021, 'Assessing kinetics and recruitment of DNA repair factors using high content screens', Cell Reports, vol. 37, no. 13, 110176. https://doi.org/10.1016/j.celrep.2021.110176

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title = "Assessing kinetics and recruitment of DNA repair factors using high content screens",

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",

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doi = "10.1016/j.celrep.2021.110176",

language = "English (US)",

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AU - Silveira, Giorgia G.

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AU - Gu, Yuchao

AU - Cosentino, Claudia

AU - Davidow, Lance S.

AU - Mata, Gadea

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AU - Salsman, Jayme

AU - Ciccia, Alberto

AU - Bae, Narkhyun

AU - Bedford, Mark T.

AU - Megias, Diego

AU - Rubin, Lee L.

AU - Efeyan, Alejo

AU - Dellaire, Graham

AU - Mostoslavsky, Raul

PY - 2021/12/28

Y1 - 2021/12/28

N2 - 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.

AB - 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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KW - high throughput

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

Abstract

Keywords

ASJC Scopus subject areas

MD Anderson CCSG core facilities

Access to Document

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