Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Vaibhav Murthy; Dalton Dacus; Monica Gamez; Changkun Hu; Sebastian O. Wendel; Jazmine Snow; Andrew Kahn; Stephen H. Walterhouse; Nicholas A. Wallace

doi:10.3791/57653

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Method Article

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

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DOI:

10.3791/57653

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June 8th, 2018

Vaibhav Murthy^*¹ , Dalton Dacus^*¹ , Monica Gamez² , Changkun Hu¹ , Sebastian O. Wendel¹ , Jazmine Snow¹ , Andrew Kahn¹ , Stephen H. Walterhouse¹ , Nicholas A. Wallace¹

¹Division of Biology, Kansas State University, ²Bristol Medical School, Translational Health Sciences, University of Bristol

^* These authors contributed equally

Summary

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

Abstract

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes.

The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Introduction

Each day, every cell in the human body is bombarded with an estimated 10,000 DNA lesions¹. This existential threat puts us at risk for mutations, oncogenesis as well as cell death. To protect genome fidelity, mammalian cells have evolved to respond to DNA damage with a complex series of protein associations and modifications. This response is organized into multiple pathways, collectively known as the DNA damage response (DDR)²^,³. The DDR consists of the accumulation of DNA repair proteins at DNA lesions, coordinated both temporally and spatially. DDR frequently induces cell cycle arres....