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

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

By: Michael J. Kruhlak¹, Arkady Celeste¹, André Nussenzweig¹

Abstract

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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.

Affiliation(s): (1) Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Book Title: Chromatin Protocols: Second Edition

Series: Methods in Molecular Biology | Volume: 523 | Pub. Date: Dec-01-2008 | Page Range: 125-140 | DOI: 10.1007/978-1-59745-190-1_9

Subject: Cell Biology

Key Words: DNA breaks - histones - chromatin - PAGFP - photoactivation - confocal microscopy - and UVA laser microirradiation

References

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