Live Cell Imaging to Study Real-Time ATM-Mediated Recruitment of DNA Repair Complexes to Sites of Ionizing Radiation-Induced DNA Damage

Methods Mol Biol. 2017:1599:287-302. doi: 10.1007/978-1-4939-6955-5_21.

Abstract

Measurements of protein recruitment and the formation of repair complexes at DNA double-strand breaks in real time provide valuable insight into the regulation of the early DNA damage response. Here, we describe the use of live cell microscopy in combination with ionizing radiation as a tool to evaluate the influence of ATM and its site-specific phosphorylation of target proteins on these processes. Recommendations are made for the preparation of the cells and the design of specialized cell chambers for the localized (and/or targeted) irradiation with charged particles at accelerator beamlines as well as the microscopic equipment and protocol to obtain high-resolution, sensitive fluorescence measurements.

Keywords: ATM; DNA damage; Double strand breaks (DSB); GFP; Live-imaging; Microscopy; Protein recruitment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins / genetics*
  • DNA Breaks, Double-Stranded / radiation effects
  • DNA Damage / genetics
  • DNA Damage / radiation effects*
  • DNA Repair / genetics
  • DNA Repair / radiation effects
  • Humans
  • Radiation, Ionizing

Substances

  • Ataxia Telangiectasia Mutated Proteins