Single‐Strand‐Specific Nuclease from the Nucleoplasm of Rye Germ Nuclei

Abstract

1. The localization of DNAase activity in rye germ nuclei revealed 48% of the total nuclear activity in the nucleoplasm and 4 % in the chromatin.2. The endonuclease present in the nucleoplasm was purified more than 300-fold as compared with the specific activity in homogenized nuclei by means of ammonium sulphate fractionation and CM-cellulose and CM-Sephadex column chromatography. The preparation gave a single band on polyacrylamide gels. Its molecular weight is 45000, pH optimum 5.4. The preparation does not exhibit activity of non-specific phosphomonoesterase and phosphodiesterase.3 . The nuclease catalyses the hydrolysis of denatured DNA and RNA to acid-soluble 5'-phosphate-terminated products and cleaves the phosphomonoester linkage of 3'AMP.4. Synthetic polyribonucleotides are hydrolysed at a rate decreasing in the order : poly(U) The endonuclease shows a high preference for single-stranded nucleic acids. It does not depolymerise double-stranded poly(U) . poly(A).6. The nuclease attacks covalently closed circular bacteriophage PM2 DNA, plasmid ColEl and plasmid pBR322 DNA, converting it initially into the open-circular (relaxed) form and subsequently into the linear form. This reaction is strongly dependent on ionic strength.7. The enzyme nicks the supercoiled DNA molecule of phage PM2 in only one place. At high enzyme concentrations several nicks in such a molecule are observed.

Single-strand-specific nucleases

“…[139] nucleases are optimally active in the range 20^50 mM NaCl, but spinach nuclease requires 50^75 mM NaCl for maximal activity [89]. Action of rye germ nuclei nuclease, on PM2 DNA, showed that it is strongly dependent on salt concentration, but the presence of high salt ( s 100 mM) results in a signi¢cant inhibition of the activity [34]. Similarly, the action of S1 nuclease, on PM2 DNA, was found to be more speci¢c at NaCl concentrations greater than 200 mM [159].…”

“…In plants, single-strand-speci¢c nucleases have been isolated from various cellular components: rye germ nuclei [34], wheat chloroplasts [35], stroma, thylakoid membrane and envelope membranes of leaf chloroplasts [36], and germinating alfalfa seeds [37]. Their presence has also been shown in the endoplasmic reticulum, Golgi apparatus, protein bodies and vacuoles of the aleurone layer of barley seeds [38], bound to chromatin in the embryo axis of germinating pea [39], cultured tobacco cells [40] and leaves of Avena [41], spinach [42] and tea [43].…”

“…For example, nucleases from wheat chloroplasts [35], wheat chloroplast stromal protein [36], rye germ ribosomes [100], nucleoplasm of rye germ nuclei [34], barley [94] and yeast [82] hydrolyze ssDNA and RNA endonucleolytically. In contrast, A. sydowii nuclease de-grades both DNA and RNA exonucleolytically in 3PC5P direction [102], whereas B. subtilis enzyme cleaves DNA exonucleolytically from the 5P-end [182].…”

An Extracellullar Nuclease fromBacillus FirmusVKPACU-1: Specificity and Mode of Action

“…[12] HPLC analysis of hydrolytic products of nuclease showed the oligonucleotides as the major end products of hydrolysis in addition to low amount of mononucleotides suggesting that the enzyme worked on endo mode of action. In addition to exo mode of action, the extracellular nucleases from wheat chloroplasts, [13] wheat chloroplast stromal protein, [14] rye germ ribosomes, [15] nucleoplasm of rye germ nuclei, [16] barley, [17] and yeast, [18] hydrolyzed ssDNA and RNA endonucleolytically. In contrast, nuclease from Flammulina velutipes exhibited endonucleolytic activity on ss and dsDNA while RNA and linear polynucleotides were degraded exonucleolytically.…”

Rye nuclease I as a tool for structural studies of tRNAs with large variable arms

“…Nuclease I from rye nucleus (Rn) was purified according to ref. (8). Activity of the enzyme preparation was 0.2 units ,ul-l (8).…”