Purification and substrate specificity of BcmI restriction endonuclease

A strain producing the new specific restriction endonuclease BcmI has been found in the Bacillus generum. The enzyme has been purified by chromatography on the blue sepharose, phosphocellulose PII, heparin sepharose. The analogous purification has been obtained when the blue sepharose has been substituted for the orange sepharose, the home produced sorbent. The BcmI enzyme has been shown by the substrate specificity definition to be an isoschizomer of the restriction endonuclease ClaI.     

Studies on the substrate specificity of the T 4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline-N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coliB_s−1 with UV-inactivated phage T4v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B_s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4v₁ is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.     

Purification of the T4 endonuclease V

A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.     

Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Photoaffinity labeling of T4 endonuclease V with a substrate containing a phenyldiazirine derivative.

T4 endonuclease V recognizes thymine photodimers in DNA duplexes and, in a two-step reaction, cleaves the glycosyl linkage of the 5'-side thymidine and the phosphodiester linkage. To determine the amino acid residues responsible for binding thymine photodimers, a photoaffinity reagent, 4-(1-azi-2,2,2-trifluoroethyl)-benzoate, was linked to the aminoalkylphosphonate of a thymine photodimer in a 14-mer duplex. The reactive substrate was treated with the enzyme under UV light (365 nm). The nascent enzyme and the modified enzyme were treated with lysyl endopeptidase, and the peptide maps were compared. Three peptides from the C terminus were found to interact with the reactive oligonucleotide to various extents. The three modified peptides were isolated and analyzed by Edman degradation. The amino acid residues Gly-133, Tyr-129, and Thr-89 were partially linked with the reactive substrate and may be involved in the binding of thymine photodimers.     

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.     

Massively parallel determination and modeling of endonuclease substrate specificity

We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.     

Determination of substrate specificity of restriction endonuclease FauI

The recognition sequence and cleavage point of restriction endonuclease FauI have been determined as 5'-CCCGC(4/6). Not being isoschisomer of any known restriction endonuclease, this enzyme may be used in genetic engineering.     

Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions

Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.     

Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-beta, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.     

Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool

An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypolynucleotides was in the order poly(dT) greater than denatured DNA greater than poly(dA) greater than poly(dA-dT) = native DNA. Neither poly(dC), poly(dG) nor poly(dC).poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear from III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I degree, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease

The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.     

Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay

Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.     

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).     

Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings.

An endonuclease was isolated from 5 days old Agropyron elongatum 8x = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme's moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn(2+) and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI > or = polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow C bond. The substrate's products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3' position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.