Superhelicity induces hypersensitivity of a human polypyrimidine . polypurine DNA sequence in the human alpha 2-alpha 1 globin intergenic region to S1 nuclease digestion--high resolution mapping of the clustered cleavage sites.
Supercoiled recombinant DNAs containing the human adult alpha-globin gene region have been probed with nuclease S1 in vitro. While agarose gel electrophoresis showed only one predominant, double-stranded cleavage generated by S1 within 6 kb of human DNA and 4 kb of pBR322 sequence, a high resolution gel analysis reveals that the unique S1-hypersensitive locus in the human adult alpha-globin gene region actually contains more than 15 authentic S1 cleavage sites closely spaced together. The mapping approach used here locates the specific S1 cleavage sites on both DNA strands at the nucleotide sequence level. Interestingly, most of these sites are mapped within a 90 bp stretch of GC-rich (66%) polypyrimidine . polypurine DNA that is located 1060 to 1150 bp upstream from alpha 1-globin gene. These results provide the first high resolution map of double-stranded S1-cleavage sites induced within a specific DNA sequence under supercoil strain. The distribution and relative cutting frequencies of these sites mapped are consistent with a slippage mechanism in which the simple repeating sequences are organized into base-mismatched duplex on supercoiled DNA.
D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004278:DNA, Superhelical / Q000235:genetics*; D004720:Endonucleases; D005796:Genes; D005914:Globins / Q000235:genetics*; D006801:Humans; D009994:Osmolar Concentration; D010957:Plasmids; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004278:DNA, Superhelical; D005914:Globins; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015719:Single-Strand Specific DNA and RNA Endonucleases