Mapping the B-A conformational transition along plasmid DNA.
A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels.
Nejedlý
Karel
K
Institute of Biophysics, Academy of Sciences of the Czech Republic Královopolská 135, CZ-61265 Brno, Czech Republic.
Chládková
Jana
J
Vorlíckov
Michaela
M
Hrabcová
Iva
I
Kypr
Jaroslav
J
D002586:Cesium / Q000494:pharmacology; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D004262:DNA Restriction Enzymes / Q000378:metabolism; D044704:DNA, A-Form / Q000737:chemistry* / Q000528:radiation effects; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010957:Plasmids / Q000737:chemistry* / Q000528:radiation effects; D014466:Ultraviolet Rays
D044704:DNA, A-Form; D002586:Cesium; D004247:DNA; D004262:DNA Restriction Enzymes