The Sse9I restriction-modification system: organization of genes and comparative analysis of proteins structures

A nucleotide sequence was established for the full-length Sporosarcina species 9D operon coding for enzymes of type II restriction-modification system Sse9I. These enzymes recognize the tetranucleotide DNA sequence 5'-AATT-3'. The operon was shown to consist of three genes that are situated with the order: sse9IC-sse9IR-sse9IM and are transcribed in the same direction. These genes encode the control protein (C.Sse9I), restriction endonuclease (R.Sse9I) and DNA-methyltransferase (M.Sse9I), respectively. A specific DNA sequence (C-box) presumably recognized by C-protein was found immediately upstream of sse9IC gene. The comparative analysis of amino acid sequences of C.Sse9I and R.Sse9I with those of relative proteins has been done. It was found that R.Sse9I revealed the most homology with the segments of R.MunI (5'-CAATTG-3') and R.EcoRI (5'-GAATTC-3'), where amino acid residues, responsible for recogniton of AATT core sequence are located. The sse9IR gene was cloned into the temperature-inducible expression vector, and recombinant Sse9I restriction endonuclease preparation was isolated.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.     

Purification and properties of recombinant DNA methyltransferase M2.<Emphasis Type="Italic">Bst</Emphasis>SE of the <Emphasis Type="Italic">Bst</Emphasis>SEI nickase-modification system

The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM.BstSEI, recognition site 5′-GAGTC-3′) includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2.BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2.BstSEI was purified by chromatography and displayed maximal activity at 55° C and pH 7.5. The enzyme modified adenine in the nickase recognition site 5′-GAGTC-3′ and was specific for 5′-GASTC-3′ substrates. The kinetic parameters of the methylation reaction were determined. The catalytic constant was 2.2 min^?1, and the Michaelis constant was 9.8 nM on T7 DNA and 5.8 μM on SAM.     

Correlation between the backbone and side chain conformations in 5′-nucleotides. The concept of a ‘rigid’ nucleotide conformation

Conformational energies of the 5′-adenosine monophosphate have been computed as a function of χ and ψ, of the torsion angles about the side-chain glycosyl C(1′)–N(9) and of the main-chain exocyclic C(4′)–C(5′) bonds by considering nonbonded, torsion, and electrostatic interactions. The two primary modes of sugar puckering, namely, C(2′)-endo and C(3′)-endo have been considered. The results indicate that there is a striking correlation between the conformations about the side-chain glyocsyl bond and the backbone C(4′)–C(5′) bond of the nucleotide unit. It is found that the anti and the Gauche–Gauche (gg), conformations about the glycosyl and the C(4′)–C(5′) bonds, respectively, are energetically the most favored conformations for 5′-adenine nucleotide irrespective of whether the puckering of the ribose is C(2′)-endo or C(3′)-endo. Calculations have also shown that the other common 5′-pyrimidine nucleotides will show similar preferences for the glycosyl and C(4′)–C(5′) bond conformations. These results are in remarkable agreement with the concept of the “rigid” nucleotide unit that has been developed from available data on mononucleotides and dinucleoside monophosphates. It is found that the conformational ‘rigidity’ in 5′-nucleotides compared with that of nucleosides is a consequence of, predominantly, the coulombic interactions between the negatively charged phosphate group and the base. The above result permits one to consider polynucleotide conformations in terms of a “rigid” C(2′)-endo or C(3′)-endo nucleotide unit with the major conformational changes being brought about by rotations about the P–O bonds linking the internucleotide phosphorus atom. IT is predicted that the anti and the gg conformations about the glycosyl and the C(4′)–C(5′) bonds would be strongly preferred in the mononucleotide components of different purine and pyrimidine coenzymes and also in the nucleotide phosphates like adenodine di- and triphosphates.     

Discrimination of three Scrophularia plants utilizing ‘Scrophularia Radix’ by DNA markers based on internal transcribed spacer (ITS) sequences

One of the medicinal materials produced by plants belonging to the genus Scrophularia, ‘Scrophularia Radix’ (SR), has been prescribed for many centuries to treat diseases such as inflammation, abscesses of carbuncles and constipation. In China, the dried root of S. ningoensis Hemsley is the source of SR. In contrast, the root of S. buergeriana is generally prescribed as SR in Korea. Studies conducted to identify the bioactive compounds in these two Scrophularia plants have revealed marked differences in the contents and concentration of the compounds they contain. However, S. ningpoensis has been indiscriminately prescribed in Korea along with S. buergeriana as SR. Furthermore, S. koraiensis has long been used in lieu of S. buergeriana as SR in Korea. Therefore, a standard or method to reliably distinguish these three species of Scrophularia is needed. Recently, we found that the differences in the nucleotide sequences of the internal transcribed spacer (ITS) of Scrophularia plants could be applied to develop DNA markers to discriminate each plant. In this study, ITS sequences of 22 samples including three types of Scrophularia plants were amplified, determined and analyzed. Based on the results of these analyses, we designed the following primer sets: Ni F (5′-TTAACCATATAGGGGCCTCG-3′) / Ni R (5′-C CCCTCTCTGTATCCCAA-3′) to amplify a 379 bp DNA marker for the identification of S. ningpoensis; Bu F (5′-TTAACC ATATCGGGGCCAAG-3′) / Bu R (5′-ATCACGACAGCAC GCGA-3′) to amplify a 491 bp DNA marker for S. buergeriana; and Ko F (5′-ATAACCATATCGGGGCCTC-3′) / Ko R (5′-TCAAGAAACGCACTATCCC-3′) to amplify a 167 bp DNA marker of S. koraiensis. Using these primer sets, we were able to efficiently identify Scrophularia plants sold as SR in the herbal market in dried and sliced states after processing as medicinal materials.     

The Fsp4HI restriction-modification system: Gene cloning, comparison of protein structures, and biochemical properties of recombinant DNA methyltransferase M.Fsp4HI

Genes coding for the Flavobacterium sp. 4H restriction-modification (RM) system, which recognizes the sequence 5′-GCNGC-3′, were cloned in Escherichia coli ER2267 and sequenced. The Fsp4HI RM system includes two genes: one for DNA methyltransferase (M.) and the other for restriction endonuclease (R.), immediately following the former in the same direction. The genes partly overlap. According to the deduced amino acid sequences, M.Fsp4HI belongs to C5 DNA methyltransferases, whereas R.Fsp4HI is only slightly similar to some restriction enzymes recognizing similar sequences. M.Fsp4HI was purified by column chromatography. The optimal conditions for the enzyme are 30°C and pH 7.5. M.Fsp4HI modifies the first cytosine in 5′-GCNGC-3′.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

Characterization of Neurospora crassa polyadenylated messenger ribonucleic acid: structure of the 5'-terminus.

Polyadenylated (poly(A)⁺) mRNA from Neurospora crassa was isolated by affinity chromatography on poly(U) Sepharose and its structure was examined. Two 5′-terminal ·cap’ structures, m⁷G(5′)ppp(5′)Ap and m⁷G(5′)ppp(5′)Gp, occurring in a relative distribution of 75 and 25% were found. No evidence was obtained for 2′-O-methylation in a nucleotide adjacent to the 5′-terminal cap.     

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

2′(3′)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di-O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)-O-(α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[¹⁴C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10^?5 M puromycin) was achieved at 9.5·10^?6 M of compound 1c and 9·10^?5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[¹⁴C]Phe to the A site of peptidyltransferase in a system containing tRNA^Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.     

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3′-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated M_r of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).     

Chlamydophila pneumoniae endonuclease IV prefers to remove mismatched 3′ ribonucleotides: Implication in proofreading mismatched 3′-terminal nucleotides in short-patch repair synthesis

DNA polymerase I (DNApolI) catalyzes DNA synthesis during Okazaki fragment maturation, base excision repair, and nucleotide excision repair. Some bacterial DNApolIs are deficient in 3′–5′ exonuclease, which is required for removing an incorrectly incorporated 3′-terminal nucleotide during DNA elongation by DNA polymerase activity. The key amino acid residues in the exonuclease center of Chlamydophila pneumoniae DNApolI (CpDNApolI) are naturally mutated, resulting in the loss of 3′–5′ exonuclease. Hence, the manner by which CpDNApolI proofreads the incorrectly incorporated nucleotide during DNA synthesis warrants clarification. C. pneumoniae encodes three 3′–5′ exonuclease activities: one endonuclease IV and two homologs of the epsilon subunit of replicative DNA polymerase III. The three proteins were biochemically characterized using single- and double-stranded DNA substrate. Among them, C. pneumoniae endonuclease IV (CpendoIV) possesses 3′–5′ exonuclease activity that prefers to remove mismatched 3′-terminal nucleotides in the nick, gap, and 3′ recess of a double-stranded DNA (dsDNA). Finally, we reconstituted the proofreading reaction of the mismatched 3′-terminal nucleotide using the dsDNA with a nick or 3′ recess as substrate. Upon proofreading of the mismatched 3′-terminal nucleotide by CpendoIV, CpDNApolI can correctly reincorporate the matched nucleotide and the nick is further sealed by DNA ligase. Based on our biochemical results, we proposed that CpendoIV was responsible for proofreading the replication errors of CpDNApolI.     

Characterization of DNA restriction and modification activities in Neisseria species

Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. NsiCI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ (EcoRV isoschizomer); NmeCI from N. meningitidis C114 and NphI from N. pharyngis C245 cleave 5′-N↓GATCN-3′ (MboI isoschizomers); NgoPII and NgoPIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ (SacII isoschizomer) and 5′-GG↓CC-3′ (HaeIII isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.     

Visualization of drug-nucleic acid interactions at atomic resolution. IV. Structure of an aminoacridine--dinucleoside monophosphate crystalline complex, 9-aminoacridine--5-iodocytidylyl (3'--5') guanosine

9-Aminoacridine forms a crystalline complex with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, space group P2₁ with $\text{a = 13.98}\text{A}\text{?}\text{, b = 30.58}\text{A}\text{?}\text{, c = 22.47}\text{A}\text{?}$ and β = 113.9 °. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by a combination of Fourier and sum-function Fourier methods. The asymmetric unit contains four 9-aminoacridine molecules, four iodoCpG molecules and 21 water molecules, a total of 245 atoms. 9-Aminoacridine demonstrates two different intercalative binding modes and, along with these, two slightly different intercalative geometries in this model system.The first of these is very nearly symmetric, the 9-amino group lying in the narrow groove of the intercalated base-paired nucleotide structure. The second shows grossly asymmetric binding to the dinucleotide, the 9-amino group lying in the wide groove of the structure. Associated with these two different intercalative binding modes is a difference in geometries in the structures. Although both structures demonstrate C3′ endo (3′–5′) C2′ endo mixed sugar puckering patterns (i.e. both cytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), with corresponding twist angles between base-pairs of about 10 °, they differ in the magnitude of the helical screw axis dislocation accompanying intercalation (Sobell et al., 1977a,b). In the pseudosymmetric intercalative structure, this value is about +0.5 Å, whereas in the asymmetric intercalative structure this value is about +2.7 Å. These conformational differences can be best described as a “sliding” of base-pairs on the intercalated acridine molecule.Although the pseudosymmetric intercalative structure can be used in 9-aminoacridine-DNA binding, the asymmetric intercalative structure cannot since this poses stereochemical difficulties in connecting neighboring sugar-phosphate chains to the intercalated dinucleotide. It is possible, however, that the asymmetric binding mode is related to the mechanism of 9-aminoacridine-induced frameshift mutagenesis (Sakore et al., 1977), and we discuss this possibility here in further detail.     

Structural Requirements forFokI-DNA Interaction and Oligodeoxyribonucleotide-instructed Cleavage

TheFokI restriction endonuclease recognizes the double-stranded (ds) 5′-GGATG-3′ site and cuts at the 9th and 13th nucleotides downstream from the 5′-3′ and 3′-5′ strands, respectively. To elucidate the interaction betweenFokI and DNA, and the effect of Mg²⁺on this interaction, we usedFokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying theFokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5′-GGATG-3′ site. When usingFokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover,FokI cleavage, when followed by step-wise filling-in ofFokI cohesive ends in the dsDNA, allowedFokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed withFokI (without Mg²⁺). Such a complex, when formed in the absence of Mg²⁺, did not accept the subsequently added Mg²⁺for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg²⁺, the half-life of the complexFokI and dsDNA was 12 minutes at 22°C. In the absence of Mg²⁺, such a complex, possessing a terminally located 5′-GGATG-3′ site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5′-GGATG-3′ site.     

Crystallographic studies of drug-nucleic acid interactions: Proflavine intercalation between the non-complementary base-pairs of cytidilyl-3′,5′-adenosine

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P4₂2₁2 with cell constants $\text{a = b = 19.38(1)}\text{A}\text{?}\text{and}\text{c = 27.10(1)}\text{A}\text{?}$. The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities.The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) Å) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) Å). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche^?-gauche^?) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche⁺ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 4₂-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids.The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.