S1 nuclease of Aspergillus oryzae.

S1 nuclease has found many uses in the analysis of the structure of nucleic acids, and more new applications, such as the mapping of splice points of early mRNAs in SV40, will undoubtedly be found.

Rapid, single-step purification of restriction endonucleases on cibacron blue F3GA-agarose.

After sonication and high-speed centrifugation, crude extracts of B. amyloliquefaciens, P. alcalifaciens, X. holicola, and B. globiggi were adsorbed on the dye Cibacron blue F3GA convalently cross-linked to agarose. The restriction endonucleases BamHI, PalI, XhoI, and BglI together with BglII were isolated by elution of the dye column with linear gradients to 0.5 M NaCl. The enzymes so purified were free of contaminating nucleic acids and other nucleases and were sufficiently concentrated for direct, specific DNA hydrolysis.

Preparation and properties of a new DNase from Aspergillus oryzae.

A DNase present in commercial preparations of Aspergillus oryzae alpha-amylase was purified 1550-fold in 25% yield by acetone precipitation and by chromatography on diethylaminoethyl- and carboxymethylcellulose. The enzyme was isolated free of contaminating RNases and DNases. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 48 000, while a molecular weight of 58 000 was determined for the single band observed upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the DNase is 9.2. The enzyme hydrolyzed only DNA with a pH optimum of 8.2 and was activated by Co2+, and to a lesser extent by Mg2+ and Mn2+. Native DNA was a better substrate than heat-denatured DNA. Enzymatic digests of calf thymus and E. coli DNA yielded oligomers of chain lengths ranging from 10 to 200, with mono- and small oligonucleotides (chain length less than 5) detected only when large (100 mg) amounts of DNA were fractionated by column chromatography on diethylaminoethyl-Sephadex A-25 in 7 M urea. The digestion products contained 5'-terminal phosphate groups and mostly adenosine at the 3' and guanosine and adenosine at the 5' ends.

DNase-sensitive sites in nucleosomes. Their relative suspectibilities depend on nuclease used.

We have used three endonucleases having different catalytic and physicochemical properties to digest HeLa nucleosomes (chromatin core particles) which had been labeled with 32P at their 5'-DNA termini. Each endonuclease nicks nucleosome DNA at the identical sites, supporting the idea that the conformation of the DNA within a nucleosome is the major factor influencing its nuclease susceptibility and indicating that nucleases can indeed yield important information as to nucleoprotein structure. On the other hand, the relaative susceptibility of a given site can differ for each nuclease, indicating that enzyme-substrate interactions unique for each enzyme influence the course of the reaction; this limits the structural information which can be obtained by using a single nuclease to study nucleoprotein structure.

S1 nuclease hydrolysis of single-stranded nucleic acids with partial double-stranded configuration.

The single-strand specific nuclease S1 from Aspergillus oryzae (EC 3.1.4.21) was purified 600-fold in 16% yield from dried mycelia. Determination of the isoelectric point of S1 nuclease as 4.3-4.4 allowed adjustment of chromatographic conditions such that the enzyme was isolated free of contaminating ribonucleases T1 and T2. S1 nuclease so purified was used for removal of single-stranded portions from the RNA of the Escherichia coli phage MS2, which has a helical content of about 65% in vitro. At 23 degrees, increasing amounts of enzyme converted the RNA to mononucleotides in about equimolar base ratios. No small intermediates of chain length 2-8 were found. At 0 degrees, MS2 RNA hydrolysis was slower and reached, in exhaustive digests, a plateau where 70% of the substrate RNA remained insoluble in 66% EtOH. With [32P]MS2 RNA, strip chart counting of 6% acrylamide-6 M urea electrophoresis patterns of such digests gave recoveries of 80-91% in the form of defined oligomer bands. On 2.5% acrylamide-0.5% agarose gels, the molecular weights of the major oligomers were found to range from 25,000 to 41,000. Similar to purified tRNAArg used as a control, these oligomers were not resistant to pancreatic RNase-RNase T1 hydrolysis at 37 degrees, and were not bound on hydroxylapatite at 50 degrees in 0.14 M sodium phosphate (pH 6.8). Melting of the oligomers gave complex profiles without a clear Tm and showed an increase in A260 of 35% at 93 degrees over that at 28 degrees. Upon formaldehyde denaturation of MS2 RNA prior to S1 nuclease hydrolysis, no resistant oligomers were found.