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Abstract
Publisher Summary This chapter deals with assay for type II restriction endonucleases using the Eseherichia coli recBC DNase and duplex circular DNA. Duplex, circular DNA is not a substrate for the recBC DNase of Escherichia coli (exonuclease V); however, linear DNA is digested by the enzyme to acid-soluble nucleotides. Therefore, the conversion of duplex, circular DNA to linear DNA by a restriction endonuclease in the presence of an excess of recBC nuclease results in the production of acid-soluble nucleotide in amounts proportional to the endonuclease activity. Properties of the assay are further described in the chapter. An estimation of endonuclease activity by this assay gave excellent quantitative agreement with results obtained by agarose gel dectrophoresis assays when either EcoR1 or Alul restriction endonucleases were utilized. EcoR1 breaks at one site on colicin E1 DNA and makes cohesive ends, whereas the Alu 1 enzyme makes roughly 50 nonstaggered breaks with this DNA. These results indicate that the exonuclease is present in excess so that a DNA molecule is essentially digested completely into acid-soluble fragments immediately upon receiving one double-stranded restriction cleavage.
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