title: UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes.
abstract: Protein-DNA interactions in mammalian cells can be analyzed at the nucleotide level of resolution by genomic sequencing techniques. The most sensitive genomic sequencing method uses the ligation-mediated polymerase chain reaction (LMPCR) for signal amplification to detect the positions of DNA modifications or strand breaks. Various probing methods are compatible with LMPCR, but dimethyl sulfate footprinting has most commonly been used. Here, we have examined the suitability of ultraviolet (UV) light as an in vivo footprinting agent to detect a wide variety of protein-DNA contacts. The distribution of the two major types of UV-induced DNA photoproducts (cyclobutane pyrimidine dimers and (6-4) photo-products) has been examined along the promoter sequences of three human genes. A comparison of UV-irradiated naked DNA and UV-irradiated cells reveals differences in the UV damage spectrum for both types of photoproducts. These differences can be either decreases or dramatic increases of photoproduct frequency. At the promoter of the c-jun gene, these differences ("photofootprints") co-localize with binding sites for two AP-1-like transcription factors, a CCAAT box binding protein, an SP-1 sequence, an NF-jun sequence, a related to serum response factor (RSRF) binding site and a sequence bound by an unknown factor. In the promoter of the gene coding for proliferating cell nuclear antigen (PCNA), photofootprints were seen at two SP-1 like sequences and two CCAAT boxes. The c-fos promoter is characterized by photofootprints at the serum response element (SRE), at the adjacent binding site for ternary complex factor (TCF), at an AP-1 site and at a binding site for a growth factor inducible protein (SIF). Photofootprints may be signatures of specific transcription factors or families of related factors since we noticed that the photofootprints seen at several common factor binding sites were similar or identical when the same site was analyzed in different genes. Photofootprints were not seen at sequences distant from transcription factor binding sites. A comparison of our UV photofootprinting data with data from experiments using other probing strategies shows that UV light has the potential to reveal all protein-DNA interactions provided there is a dipyrimidine sequence on either DNA strand within a factor binding site. The simplicity of using this probing agent together with its specificity for detecting a large variety of different factors should make UV light a generally useful tool for in vivo footprinting studies.
authors.affiliation: Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
authors.forename: S
authors.initials: S
authors.lastname: Tornaletti
authors.forename: G P
authors.initials: GP
authors.lastname: Pfeifer
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D014157:Transcription Factors; D004247:DNA