Sequence preferences in cleavage of dsDNA and ssDNA by the extracellular XXXX endonuclease.

@entity71843 @entity5134 @entity1133 @entity3733 @entity291
The preferred cleavage sites in dsDNA and ssDNA for the extracellular @entity5134 endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioactively or fluorescent dye labeled primers. ssDNA of identical sequence to one of the fluorescent dye labeled duplex strands was prepared by affinity chromatography. Cleavage experiments carried out under single hit conditions demonstrate that the enzyme shows preferences for GC-rich regions in dsDNA, in particular d(G).d(C)-tracts, and avoids cleavage of d(A).d(T)-tracts. There is a correlation between cleavage at a given position in one strand with cleavage at the same position in the other strand of the duplex. ssDNA cleavage occurs at somewhat different preferred sites than observed in dsDNA. On dsDNA, the Serratia nuclease produces a very different cleavage pattern compared to @entity291 pancreatic @entity71843 , with the notable exception that both enzymes avoid d(A).d(T)-tracts. In general, the Serratia nuclease compared to @entity71843 is a slightly more nonspecific endonuclease that attacks a particular substrate more evenly under standard reaction conditions. At high ionic strength or in the presence of @entity3733 , it becomes more nonspecific. Addition of @entity1133 , however, makes the enzyme more selective than observed under standard conditions. From these results which were confirmed by the results of cleavage experiments with synthetic oligodeoxynucleotides, we conclude that the Serratia nuclease like @entity71843 is sensitive to global features of the DNA, for example, the width of the minor groove. In addition, localized sequence-dependent interactions between substrate and nuclease determine whether a site is cleaved preferentially. Some of these interactions seem to be the same for ds- and ssDNA.
