Protecting recognition sequences on DNA by a cleavage-deficient restriction endonuclease.
This report describes the use of a biochemical tool that has been developed to aid in the manipulation of DNA. A DNA binding-proficient and cleavage-deficient BamHI mutant protein, E113K, was used in vitro to protect its recognition sequence (5'-GGATCC-3') against the catalytic action of site-specific endonuclease, exonuclease and methylase. In vitro conditions are reported here in which the E113K protein protects BamHI sites (5'-GGATCC-3') from cleavage by BamHI endonuclease or Sau3AI endonuclease (5'-GATC-3'); protects a neighboring restriction site 5'-CCCGGG-3' from SmaI endonuclease digestion; blocks methylation of 5'-GGATCC-3' by Dam methylase (5'-GATC-3'); and blocks Bal31 exonuclease progression at a BamHI site. The Bal31 procedure could be used to generate unidirectional deletions of a DNA fragment. The use of mutant endonucleases that are binding-proficient and cleavage-deficient to shield DNA from nuclease digestion or methylase modification expands the repertoire of methods to manipulate DNA in vitro.
New England Biolabs, Beverly, MA 01915.
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D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
D004247:DNA; D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases