Interaction of the periplasmic dG-selective Streptomyces antibioticus nuclease with oligodeoxynucleotide substrates.
The interaction of a periplasmic nuclease, isolated from Streptomyces antibioticus, with several oligodeoxynucleotide substrates has been studied. Double-stranded oligonucleotides that contain sequences of four or more consecutive deoxyguanosine residues are preferentially hydrolyzed, with the strongest cutting site occurring at GGG decreases G. The enzyme does not hydrolyze these sequences in single-stranded DNA. However the sequence selectivity of the nuclease is far from absolute. Other sequences can also be cut, albeit more poorly, and differences in cutting rates are observed for runs of dG bases that differ in their flanking sequences. An oligonucleotide, thirty-six bases in length, that contains a central run of five dG bases has been used to evaluate the importance of the individual deoxyguanosines in recognition and cleavage. With this oligonucleotide cutting takes place at GG[symbol: see text]G decreases G[symbol: see text]G (decreases, most prominent cut; [symbol: see text], less prominent cuts). 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 decreases), were of most importance in the determination of the nuclease DNA cleavage selectivity. Of these the inner starred dG was the most critical. The use of 5-methyldeoxycytidine also showed that the dC, base paired to this critical dG, influenced cleavage specificity. The overall pattern of results seen with the base analogues suggested that the nuclease interacted 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 radicals, 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+.
Cal
S
S
Departamento de Biologia Funcional, Universidad de Oviedo, Spain.
Nicieza
R G
RG
Connolly
B A
BA
Sánchez
J
J
D001483:Base Sequence; D018983:DNA Footprinting; D003849:Deoxyguanosine / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004720:Endonucleases; D003849:Deoxyguanosine