Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Xiangduo Kong; Gladys M.S. Cruz; Bárbara A. Silva; Nicole M. Wakida; Nima Khatibzadeh; Michael W. Berns; Kyoko Yokomori

doi:10.3791/56213

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Method Article

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

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DOI:

10.3791/56213

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January 31st, 2018

Xiangduo Kong^*¹ , Gladys M.S. Cruz^*² , Bárbara A. Silva² , Nicole M. Wakida² , Nima Khatibzadeh² , Michael W. Berns²^,³^,⁴ , Kyoko Yokomori¹

¹Department of Biological Chemistry, School of Medicine, University of California, Irvine, ²Beckman Laser Institute and Medical Clinic, University of California, Irvine, ³Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, ⁴Department of Biomedical Engineering and Surgery, University of California, Irvine

^* These authors contributed equally

Summary

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Abstract

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Introduction

In Vivo DNA Damage Signaling is Not Well Understood
In vivo, DNA is complexed with histones and other factors to form chromatin fibers. Regulation of chromatin structure is of paramount importance for DNA metabolism. For example, the histone variant H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other kinases following double-strand breaks (DSB) induction, and is important for DSB damage signal amplification as well as providing a docking site for other factors. Spreading of damage signaling and repair pathway choice appear to be critically influenced by local chromatin structure at damage sites