Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.     

A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt

The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the α- and β-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in β-hordothionin and Cys29-Arg30 of α-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P₂ position, not those immediately adjacent to the susceptible bonds. The P₂ amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P₁-P₁′ in the general sequence NH₂—P₂-P₁-P₁′—COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P₂. The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity.     

Control of 5′,5′-Dinucleoside Triphosphate Catabolism by APH1, a Saccharomyces cerevisiae Analog of Human FHIT

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P¹,P³-triphosphate (Ap₃A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap₃A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap₃A-hydrolyzing activity and a 30-fold-elevated Ap₃N concentration.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15′-oxygenase (BCO1) and the β-carotene-9′,10′-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15′-apocarotenoids, C₂₀), whereas eccentric cleavage by BCO2 produces long-chain (>C₂₀) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1^−/−, Bco2^−/−, and Bco1^−/−Bco2^−/− double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10′-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

Comparative specificity of microbial acid proteinases for synthetic peptides. II. Effect of secondary interaction

To investigate the effect of “secondary interaction” on hydrolysis by various acid proteinases from molds and yeasts, synthetic peptides

amino acid residues) were used as substrates. Pepsin was used for the comparative study. These peptides were split at the peptide bonds indicated by the arrows, permitting examination of the effect of residue X distant by two or three amino acid residues from the hydrolytic site in the peptides. According to the system of Schechter and Berger (Biochem. Biophys. Res. Commun. 27; 157, 1967), the amino acid residues in peptide substrates were numbered P₁, P₂, etc. toward the N-terminal direction from the site of hydrolysis, and P₁′, P₂′, etc. toward the C-terminal direction. The results indicated that hydrolysis by these microbial enzymes is affected by at least six amino acid residues (P₁-P₃ and P₁′-P₃′) in peptide substrates, as is seen with pepsin. Elongation of the peptide chain with suitable amino acid residues from P₁ to P₂ or P₃ and from P₁′ to P₂′ or P₃′ in peptide substrates resulted in much or less increase of hydrolysis depending upon the species of the enzyme producers.     

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C,a Key Regulator of Digestive Enzyme Activation

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S₆-S₅′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P₁, P₄, and P₂′ positions of CTRC, although acidic P₂′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P₆ position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.     

Recognition of ribonuclease A by 3'-5'-pyrophosphate-linked dinucleotide inhibitors: a molecular dynamics/continuum electrostatics analysis

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5′-phospho-2′-deoxyuridine-3-pyrophosphate (P→5)-adenosine-3-phosphate (pdUppA-3′-p). The 3′,5′-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B₂ and the placement of the 5′-β-phosphate of the adenylate (instead of the α-phosphate) at subsite P₁ where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3′-p and the related weaker inhibitor dUppA, which lacks the 3′ and 5′ terminal phosphate groups of pdUppA-3′-p. The simulations show that the adenylate 5′-β-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P₁ and B₂. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3′-p varies between −7.9 kcal/mol and −2.8 kcal/mol for a range of protein/ligand dielectric constants (ε_p) 2–20, in good agreement with the experimental value (−3.6 kcal/mol); the agreement becomes exact with ε_p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3′-p make increased interactions with residues Lys⁷ and Lys⁶⁶ of the more remote sites P₂ and P₀, and His¹¹⁹ of site P₁.     

Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp⁹⁷ is proposed to interact with the P1′ amino acid of its substrates, OmpT variants with variations at Asp⁹⁷ were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1′). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg↓motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

In Vitro Synergistic Antioxidant Activity and Identification of Antioxidant Components from Astragalus membranaceus and Paeonia lactiflora

Many traditionally used herbs demonstrate significantly better pharmacological effects when used in combination than when used alone. However, the mechanism underlying this synergism is still poorly understood. This study aimed to investigate the synergistic antioxidant activity of Astragalus membranaceus (AME) and Paeonia Lactiflora (PL), and identify the potential antioxidant components by 1,1-diphenyl-2-picrylhydrazine (DPPH) radical spiking test followed by a high performance liquid chromatography separation combined with diode array detection and tandem mass spectrometry analysis (DPPH-HPLC-DAD-MS/MS). Eight AME-PL combined extracts (E₁–E₈) were prepared based on bioactivity-guided fractionation. Among them, E₁ exhibited the strongest synergistic effect in scavenging DPPH radicals and reducing ferric ions (P<0.05). Moreover, E₁ presented strong cytoprotection against H₂O₂-induced oxidative damage in MRC-5 cells by suppressing the decrease of the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activities. A strong correlation between the increment of total phenolic/flavonoid and synergistic antioxidant activity, especially between the increment of total flavonoid and the increase in ferric reducing power was observed. Finally, seven antioxidant substances were identified in E₁ as oxypaeoniflora, catechin, calycosin-7-O-β-D-glucopyranoside, fomononetin-7-O-β-D-glucopyranoside, 9,10-dimethoxy-pterocarpan-3-O-β-D-glucopyranoside, quercetin and 2′-dihydroxy-3′,4′-dimethyl-isoflavan-7-O-β-D-glucopyranoside.     

Degradation of the γ-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4′-Ethoxy-3′-Methoxyphenyl)-2-(4″-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4′-ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [¹⁴C]lignin to ¹⁴CO₂. 1-(4′-Ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by α, β cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under ¹⁸O₂, ¹⁸O was incorporated into the β position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

The C nucleotide at the mature 5′ end of the Escherichia coli proline tRNAs is required for the RNase E cleavage specificity at the 3′ terminus as well as functionality

Proline tRNA 3′-maturation in Escherichia coli occurs through a one-step RNase E endonucleolytic cleavage immediately after the CCA determinant. This processing pathway is distinct from the 3′-end maturation of the other tRNAs by avoiding the widespread use of 3′ → 5′ exonucleolytic processing, 3′-polyadenylation and subsequent degradation. Here, we show that the cytosine (C) at the mature 5′-terminus of the proK and proL tRNAs is required for both the RNase E cleavage immediately after the CCA determinant and their functionality. Thus, changing the C nucleotide at the mature 5′-terminus of the proL and proK tRNAs to the more common G nucleotide led to RNase E cleavages 1–4 nucleotides downstream of the CCA determinant. Furthermore, the 5′-modified mutant tRNAs required RNase T and RNase PH for their 3′-maturation and became substrates for polyadenylation and degradation. Strikingly, the aminoacylation of the 5′-modified proline tRNAs was blocked due to the change in the recognition element for prolyl-tRNA-synthetase. An analogous modification of the pheV 5′-mature terminus from G to C nucleotide did not support cell viability. This result provides additional support for the importance of first nucleotide of the mature tRNAs in their processing and functionality.     

Mutations Affecting the Ability of the Escherichia coli UmuD′ Protein To Participate in SOS Mutagenesis

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190–7194, 1990). To identify elements of the UmuD′ protein necessary for its role in translesion synthesis, we began with umuD′, a modified form of the umuD gene that directly encodes the UmuD′ protein, and obtained missense umuD′ mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD′₂ homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded β-sheet and appears to interfere with UmuD′₂ homodimer formation indirectly by affecting the structure of the UmuD′ monomer. When expressed from a multicopy plasmid, the L40R umuD′ mutant gene exhibited a dominant negative effect on a chromosomal umuD⁺ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD′ function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal β-strand and results in a mutant UmuD′ protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD⁺ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD′₂ homodimer, which are mobile in solution.     

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH₃, 2′-H or 2′-F modification at the canonical (c₀) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c₀ site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH₃ suppressed processing at the major aberrant m₋₁ site; instead, the m₊₁ (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c₀ and m₊₁ sites had different pH profiles, with a higher Mg²⁺ requirement for c₀ versus m₊₁ cleavage. The strong catalytic defect for LNA and 2′-OCH₃ supports a model where the extra methylene (LNA) or methyl group (2′-OCH₃) causes a steric interference with a nearby bound catalytic Mg²⁺ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.     

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.     

Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

Resection of the 5′-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5′ ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60–70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.     

Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, K_m and V_max were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.     

Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage

Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3′-OH and 5′-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3′-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3′-S-phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3′-S-phosphorothiolate-modified ptRNA carrying a 7 nt 5′-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5′-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn²⁺ or Cd²⁺. To suppress aberrant cleavage, we also constructed a 3′-S-phosphorothiolate-modified ptRNA with a 1 nt 5′-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3′-S-phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.