Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation. Analogies with the biochemical processes described in late steps of eukaryotic apoptosis.

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.

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+.

A novel exocytoplasmic endonuclease from Streptomyces antibioticus.

A new exocytoplasmic, nutritionally controlled endodeoxyribonuclease (EC 3.1.21.-) was purified to homogeneity from Streptomyces antibioticus. The enzyme showed an apparent molecular mass of 29 kDa (being active in the monomeric form) and a pI of approximately 7.8. The nuclease hydrolysed endonucleolytically double-stranded circular and linear DNA. The enzyme makes nicks in one strand of the DNA in G-rich regions, leaving either 5' or 3' short, single-stranded overhangs with 3'-hydroxy and 5'-phosphate termini. Breaks in the DNA occur when two nicks in opposite strands are close together. The enzyme had an optimum pH of 7.5 and an absolute requirement for bivalent cations and > or = 100 mM NaCl in the reaction buffer. Activity was greatly diminished in the presence of phosphate, Hg2+ or iodoacetate and was stimulated by dimethyl sulphoxide. Single-stranded DNA was a much poorer substrate than double-stranded DNA. The nuclease hydrolyses sequences of three or preferably more (dG).(dC) tracts in the DNA. The initial specificity shifts to other sequences (including sequences shorter than those initially hydrolysed) during the course of the reaction, giving the changing pattern of bands observed in agarose gels. 5-Methylcytosine-hemimethylated DNA is not hydrolysed by the nuclease. The properties of this novel enzyme suggest a relationship with class II restriction endonucleases and also with some eukaryotic nucleases.