Zero-length protein-nucleic acid crosslinking by radical-generating coordination complexes as a probe for analysis of protein-DNA interactions in vitro and in vivo.
Redox-active coordination complexes such as 1,10-phenanthroline-Cu(II) (OP-Cu) and bleomycin-Fe(III) are commonly used as "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we report that under certain conditions these complexes may crosslink proteins to nucleic acids. In vitro experiments suggest that proteins are crosslinked to DNA by a mechanism similar to dimethyl sulfate-induced crosslinking. Furthermore, we demonstrate that the OP-Cu complex can generate protein-DNA crosslinks in mammalian cells in vivo. By combining the OP-Cu crosslinking and a "protein shadow" hybridization assay we identify proteins interacting with DNA in isolated pea chloroplasts and show that this methodology can be applied to detect DNA-binding proteins on specific DNA sequences either in vitro or in vivo.
Gavin
I M
IM
W. A. Engelhardt Institute of Molecular Biology, Academy of Sciences of Russia, Vavilova, 32, Moscow B-334, 117984, Russia.
Melnik
S M
SM
Yurina
N P
NP
Khabarova
M I
MI
Bavykin
S G
SG
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D001761:Bleomycin / Q000031:analogs & derivatives / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005609:Free Radicals; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D009696:Nucleic Acids / Q000737:chemistry*; D010618:Phenanthrolines / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012265:Ribonucleotides / Q000737:chemistry
D002843:Chromatin; D003432:Cross-Linking Reagents; D005609:Free Radicals; D006657:Histones; D009696:Nucleic Acids; D010618:Phenanthrolines; D011506:Proteins; D012265:Ribonucleotides; C023503:iron bleomycin; D001761:Bleomycin; C014741:bis(1,10-phenanthroline)copper(2+) ion; D004247:DNA