SEQUENCE PREFERENCES IN CLEAVAGE OF DSDNA AND SSDNA BY THE EXTRACELLULAR SERRATIA-MARCESCENS ENDONUCLEASE

The preferred cleavage sites in dsDNA and ssDNA for the extracellular Serratia marcescens endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioacti vely or fluorescent dye labeled primers. ssDNA of identical sequence t o one of the fluorescent dye labeled duplex strands was prepared by af finity chromatography. Cleavage experiments carried out under single h it conditions demonstrate that the enzyme shows preferences for GC-ric h regions in dsDNA, in particular d(G). d(C)-tracts, and avoids cleava ge 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 differe nt preferred sites than observed in dsDNA. On dsDNA, the Serratia nucl ease produces a Very different cleavage pattern compared to bovine pan creatic DNase I, with the notable exception that both enzymes avoid d( A). d(T)-tracts. In general, the Serratia nuclease compared to DNase I is a slightly more nonspecific endonuclease that attacks a particular substrate more evenly under standard reaction conditions. At high ion ic strength or in the presence of DMSO, it becomes more nonspecific. A ddition of urea, however, makes the enzyme more selective than observe d under standard conditions. From these results which were confirmed b y the results of cleavage experiments with synthetic oligodeoxynucleot ides, we conclude that the Serratia nuclease like DNase I is sensitive to global features of the DNA, for example, the width of the minor gr oove. In addition, localized sequence-dependent interactions between s ubstrate and nuclease determine whether a site is cleaved preferential ly. Some of these interactions seem to be the same for ds- and ssDNA.