Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: specific reactivity with short A-tracts.
We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G). The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene. The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent. In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity. In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts. Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine. Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C. However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost. Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures. The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA. This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts. The relevance of these results to current models of A-tract structure and DNA bending is discussed. Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.
McCarthy
J G
JG
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Williams
L D
LD
Rich
A
A
D000225:Adenine / Q000378:metabolism; D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA; D015342:DNA Probes; D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D005561:Formates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011196:Potassium Permanganate / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D013696:Temperature; D013941:Thymine / Q000378:metabolism; D013997:Time Factors; D001618:beta-Lactamases / Q000235:genetics
D015342:DNA Probes; D005561:Formates; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D011196:Potassium Permanganate; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D001618:beta-Lactamases; D000225:Adenine; D004047:Diethyl Pyrocarbonate; D013941:Thymine