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Monitoring DNA Breaks in Optically Highlighted Chromatin in Living Cells by Laser Scanning Confocal Microscopy

  • Michael J. Kruhlak
  • Arkady Celeste
  • André Nussenzweig
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 523)

Abstract

The recognition and repair of DNA lesions occurs within a chromatin environment. Genetically tagging fluorescent proteins to DNA damage response proteins has provided spatial and temporal details concerning the establishment of biochemical subnuclear regions geared toward metabolizing genomic lesions. A specific marker for chromatin regions containing DNA breaks is required to study the initial dynamic structural changes in chromatin when DNA breaks occur. Here we present the experimental protocols used to investigate the dynamics of chromatin structure immediately after the simultaneous photoactivation of PAGFP-tagged core histone H2B and introduction of DNA breaks using UVA laser microirradiation on a laser scanning confocal microscope.

Key words

DNA breaks histones chromatin PAGFP photoactivation confocal microscopy and UVA laser microirradiation 

Notes

Acknowledgments

We thank George Patterson (NICHD/NIH) for the generous gift of the PAGFP construct and helpful technical assistance. We are grateful to Tom Misteli (NCI/NIH) for providing the H2B-GFP construct.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Michael J. Kruhlak
    • 1
  • Arkady Celeste
    • 1
  • André Nussenzweig
    • 1
  1. 1.Experimental Immunology BranchNational Cancer Institute, National Institutes of HealthBethesdaUSA

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