Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex.
Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology
D011089:Polydeoxyribonucleotides; C050366:poly(dG-dA)n.poly(dC-dT)n; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases