Site-directed DNA crosslinking of large multisubunit protein-DNA complexes.
Several methods have been developed to site-specifically incorporate photoreactive nucleotide analogs into DNA for the purpose of identifying the proteins and their domains that are in contact with particular regions of DNA. The synthesis of several deoxynucleotide analogs that have a photoreactive group tethered to the nucleotide base and the incorporation of these analogs into DNA are described. In a second approach, oligonucleotide with a photoreactive group attached to the phosphate backbone is chemically synthesized. The photoreactive oligonucleotide is then enzymatically incorporated into DNA by annealing it to a complementary DNA template and extending with DNA polymerase. Both approaches have been effectively used to map protein-DNA interactions in large multisubunit complexes such as the eukaryotic transcription or ATP-dependent chromatin remodeling complexes. Not only do these techniques map the binding sites of the various subunits in these complexes, but when coupled with peptide mapping also determine the protein domain that is in close proximity to the different DNA sites. The strength of these techniques is the ability to scan a large number of potential sites by making combinations of different DNA probes and is facilitated by using an immobilized DNA template for synthesis.
Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901-4413, USA.
Jim
J
Persinger
Blaine
B
Bartholomew
D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes / Q000138:chemical synthesis; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010449:Peptide Mapping; D019920:Photoaffinity Labels; D021122:Protein Subunits / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D015342:DNA Probes; D009711:Nucleotides; D019920:Photoaffinity Labels; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA