Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA.
Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.
Tsen
Hua
H
Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, PO Box 830688, Richardson, TX 75083, USA.
Levene
Stephen D
SD
D001483:Base Sequence; D045566:DNA, Cruciform; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D006868:Hydrolysis; D017665:Hydroxyl Radical; D008962:Models, Theoretical; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D045566:DNA, Cruciform; D004278:DNA, Superhelical; D017665:Hydroxyl Radical; D004720:Endonucleases