![]() ![]() AarI does not cleave λ DNA or any other substrate completely. ![]() Column samples (3 µl) were added to the reaction mixture, incubated for 15 min at 37☌ and aliquots from each solution were then electrophoresed on a 0.8% agarose gel. The restriction endonuclease activity was assayed by incubation of 1 µg λ DNA in 50 µl of AarI reaction mixture: 10 mM Bis-Tris propane–HCl pH 6.5, 100 mM KCl, 10 mM MgCl 2 and 0.1 mg/ml BSA. Many AarI sites are not cleaved completely under standard restriction reaction conditions and AarI activity can be stimulated by an oligonucleotide duplex containing the recognition sequence of the enzyme. We report here the isolation and characterization of a new restriction endonuclease AarI from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic heptanucleotide sequence 5′-CACCTGC(N) 4/8-3′ and cleaves DNA 4 and 8 nt away from the target duplex producing a four base 5′-protruding end. Many type II restriction endonucleases do not conform to the standard definition of type II enzymes and, therefore, they were divided into subdivisions, so-called types IIS, IIE, IIF, IIT, IIG, IIB and IIM ( 2). The main criterion for classifying a restriction endonuclease as a type II enzyme is that it cleaves specifically within or close to its recognition sequence and that it does not require ATP hydrolysis for its nucleolytic activity. Type II restriction endonucleases recognize short, usually palindromic, sequences of 4–8 bp and, in the presence of Mg 2+, cleave DNA within or in close proximity to the target sequence. More than 3000 type II restriction endonucleases have been discovered but only 231 are examples of enzymes with different and unique specificities (prototypes) ( 1). ![]()
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