Co-localization of DNA repair proteins with UV-induced DNA damage in locally irradiated cells.
This chapter describes a technique in which indirect immunofluorescence is applied to visualize the process of nucleotide excision repair (NER) at the site of locally induced damage in DNA. UV-irradiation of cells through an isopore polycarbonate membrane filter generates cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP) on a subnuclear area, which corresponds to the size of a pore on the membrane. Specific antibodies to CPD and 6-4PP define the damaged spot. The NER components co-localize at the damaged-DNA subnuclear spot, where the proteins are stained with the appropriate fluorescent antibodies. This relatively simple and affordable method facilitates the examination of the sequential assembly of NER proteins in the chromatin-embedded DNA photoproducts. The method also enhances the identification of repair auxiliary proteins and complexes, such as ubiquitin E3 ligases, involved in the initiation of NER on non-transcribed DNA.
Department of Microbiology and Molecular Genetics, Hillman Cancer Center, University of Pittsburgh School of Medicine, and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
Jennifer
J
Guerrero-Santoro
Arthur S
AS
Levine
Vesna
V
Rapić-Otrin
D002021:Buffers; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D003902:Detergents; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D021381:Protein Transport / Q000528:radiation effects; D014466:Ultraviolet Rays
D002021:Buffers; D003902:Detergents; D045643:DNA Repair Enzymes