Footprinting with UV irradiation and LMPCR.
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 ligation-mediated polymerase chain reaction (LMPCR) for signal amplification. Several footprinting methods are compatible with LMPCR. Here we describe in detail the use of UV irradiation for in vivo footprinting. The distribution of the two major types of UV-induced DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] can be analyzed by LMPCR and a wide variety of protein-DNA contacts can be detected by analyzing both photoproducts. A comparison of UV photofootprinting data with data from experiments using other probing techniques shows that UV light has the potential to reveal all protein-DNA interactions provided that there is a dipyrimidine sequence on either DNA strand within a factor binding site. The simplicity and nondisruptiveness of this probing method together with its ability to detect a large number of different transcription factors should make UV light a generally useful tool for in vivo footprinting. We provide protocols for UV footprinting and LMPCR.
Pfeifer
G P
GP
Department of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA. gpfeifer@smtplink.coh.orgp5
Tornaletti
S
S
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007202:Indicators and Reagents; D008322:Mammals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011740:Pyrimidine Dimers / Q000032:analysis; D014466:Ultraviolet Rays
D017931:DNA Primers; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D011740:Pyrimidine Dimers; D004247:DNA