PubMed · 13738340
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R J PRZYBYLSKI. 1961. Electron microscope autoradiography.. https://doi.org/10.1016/0014-4827(61)90269-5
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Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.
The unscheduled DNA synthesis (UDS) assay measures a cell's ability to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique in living cells by creating 6-4 photoproducts and pyrimidine dimers using UVC irradiation, then allowing for their repair. Repair is quantified by the amount of radioactive thymidine incorporated after this insult, and the length of time allowed for this incorporation is specific for repair of particular lesions. Radioactivity is evaluated by grain counting after autoradiography. The results are used to diagnosis repair-deficient disorders clinically and provide a basis for investigation of repair deficiency in human tissues or tumors. At the present time, no other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation, as discussed in Chapter 28, is not an equivalent technique, as it specifically measures transcription-coupled repair at active genes, a subset of total NER.
Human nicotinic acetylcholine receptor (nAChR) alpha7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows alpha7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled alpha-bungarotoxin (I-Bgt) binding sites corresponding to human alpha7 (halpha7)-nAChRs on the surface of most cells. I-Bgt binds to halpha7-nAChRs in membrane fractions with a typical apparent K(D) value of approximately 5 nM and B(max) value of approximately 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed halpha7-nAChRs is evident as rapid, transient inward current responses to (-)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to approximately 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing alpha3 or alpha4 subunits (K(D) values of about 100 or 10 pM, respectively). halpha7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (K(D) value of approximately 0.6 nM). Studies of several forms of native or heterologously expressed rat or human alpha7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and alpha-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine approximately cytisine > carbamylcholine approximately D-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human alpha7-nAChRs that exhibit ligand binding and functional properties like native alpha7-nAChRs and that epibatdine is useful as a probe for human alpha7-nAChRs.