A method for detecting protein-DNA interactions at sites of chromatin replication.
Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [alpha-32P] dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins--marked by short covalently linked radioactive DNA tags--are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.
Blanco
J
J
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Kimura
H
H
Mueller
G C
GC
D002843:Chromatin / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008836:Micrococcal Nuclease; D009698:Nucleoproteins / Q000378:metabolism*; D014466:Ultraviolet Rays
D002843:Chromatin; D003432:Cross-Linking Reagents; D009698:Nucleoproteins; D004247:DNA; D008836:Micrococcal Nuclease