INTERACTION OF THE PERIPLASMIC DG-SELECTIVE STREPTOMYCES-ANTIBIOTICUSNUCLEASE WITH OLIGODEOXYNUCLEOTIDE SUBSTRATES

The interaction of a periplasmic nuclease, isolated from Streptomyces antibioticus, with several oligodeoxynucleotide substrates has been st udied, Double-stranded oligonucleotides that contain sequences of four or more consecutive deoxyguanosine residues are preferentially hydrol yzed, with the strongest cutting site occurring at GGG down arrow G, T he enzyme does not hydrolyze these sequences in single-stranded DNA. H owever the sequence selectivity of the nuclease is far from absolute, Other sequences can also be cut, albeit more poorly, and differences i n cutting rates are observed for runs of dG bases that differ in their flanking sequences. An oligonucleotide, thirty-six bases in length, t hat contains a central run of five dG bases has been used to evaluate the importance of the individual deoxyguanosines in recognition and cl eavage, With this oligonucleotide cutting takes place at GG del G down arrow G del G (down arrow, most prominent cut; del, less prominent cu ts), The use of dG base analogues revealed that two bases, one and two steps removed from the cleavage site in the 5' direction (G*GG down arrow), were of most importance in the determination of the nuclease D NA cleavage selectivity, Of these the inner stat-red dG was the most c ritical, The use of 5-methyldeoxycytidine also showed that the dC, bas e paired to this critical dG, influenced cleavage specificity. The ove rall pattern of results seen with the base analogues suggested that th e nuclease intel acted with both strands of the DNA and also contacted the nucleic acid in both the major and minor grooves, Gel retardation analysis together with footprinting experiments using hydroxyl radica ls, dimethyl sulfate, and ethylnitrosourea indicated that the nuclease does not form a tight and specific complex with sequences containing dG runs, at least in the absence of the essential co-factor, Mg2+.