A versatile in vivo footprinting technique using 1,10-phenanthroline-copper complex to study important cellular processes.
A number of reagents have been used to define the sequence-specific protein-DNA contacts by footprinting analysis. We report a new in vivo technique using the complex of 1,10-phenanthroline and copper [(OP(2))Cu] as a probe to study various intracellular DNA-protein interactions in whole cells. The versatility of the protocol is demonstrated by applying the technique to address various processes. The protocol is applied to (i) detect structural alterations in DNA as a result of single base substitution, (ii) footprint site-specific DNA-binding proteins, (iii) analyze promoter occupancy by RNA polymerase and (iv) analyze molecular interactions during transcription initiation. The results demonstrate that in vivo (OP)(2)Cu probing is a useful tool in studying important cellular processes involving DNA-protein interactions and has potential applications in post-genomic research.
Basak
S
S
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Nagaraja
V
V
D001483:Base Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D010618:Phenanthrolines / Q000378:metabolism* / Q000494:pharmacology; D017354:Point Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000235:genetics
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010618:Phenanthrolines; D003300:Copper; D004247:DNA; C072049:DNA modification methylase KpnI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D012321:DNA-Directed RNA Polymerases; C025205:1,10-phenanthroline