Monitoring DNA breaks in optically highlighted chromatin in living cells by laser scanning confocal microscopy.
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.
Kruhlak
Michael J
MJ
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Celeste
Arkady
A
Nussenzweig
André
A
D000818:Animals; D002138:Calibration; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D053960:DNA Breaks / Q000528:radiation effects*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism / Q000528:radiation effects*; D006657:Histones / Q000378:metabolism; D007834:Lasers; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D055620:Optical Phenomena; D010766:Phosphorylation / Q000528:radiation effects; D013194:Staining and Labeling; D014162:Transfection; D014466:Ultraviolet Rays
D002843:Chromatin; D006657:Histones; C475961:gamma-H2AX protein, mouse