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 s...

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Bibliographische Detailangaben
Veröffentlicht in:	BioTechniques. - 1988. - 15(1993), 2 vom: 01. Aug., Seite 310-5
1. Verfasser:	Xu, S Y (VerfasserIn)
Weitere Verfasser:	Schildkraut, I
Format:	Aufsatz
Sprache:	English
Veröffentlicht:	1993
Zugriff auf das übergeordnete Werk:	BioTechniques
Schlagworte:	Journal Article Research Support, Non-U.S. Gov't DNA 9007-49-2 Methyltransferases EC 2.1.1.- Dam methyltransferase EC 2.1.1.72 Site-Specific DNA-Methyltransferase (Adenine-Specific) Endodeoxyribonucleases mehr... EC 3.1.- exonuclease Bal 31 EC 3.1.11.- DNA Restriction Enzymes EC 3.1.21.- Deoxyribonuclease BamHI CCCGGG-specific type II deoxyribonucleases EC 3.1.21.4 Deoxyribonucleases, Type II Site-Specific GATC-specific type II deoxyribonucleases

Beschreibung
Zusammenfassung:	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
Beschreibung:	Date Completed 20.10.1993 Date Revised 29.08.2008 published: Print Citation Status MEDLINE
ISSN:	1940-9818