Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences.

RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).     

Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase.

The original cDNA sequence reported for the murine DNA methyltransferase (MTase) was not full length. Recently, additional cDNA sequences have been reported that lie upstream of the original and contain an extended open reading frame with three additional ATGs in frame with the coding region [Tucker et al . (1996) Proc. Natl. Acad. Sci. USA , 93, 12920-12925; Yoder et al . (1996) J. Biol. Chem . 271, 31092-31097]. Genomic DNA upstream of this ATG contains two more ATGs in frame and no obvious splice site. We have constructed, and expressed in baculovirus, MTase clones that begin at each of these four ATGs and examined their properties. Constructs beginning with any of the first three ATGs as their initiator methionines give a predominant DNA MTase band of approximately 185 kDa on SDS-PAGE corresponding to translational initiation at the third ATG. The fourth ATG construct gives a much smaller protein band of 173 kDa. The 185 kDa protein was purified by HPLC, characterized by mass spectrometry and has a measured molecular mass of 184 +/- 0.5 kDa. All of these MTases were functional in vitro and steady state kinetic analysis showed that the recombinant proteins exhibit similar kinetic properties irrespective of their length. The homogeneous recombinant enzyme from the fourth ATG construct shows a 2.5-fold preference for a hemi-methylated DNA substrate as compared to an unmethylated substrate, whereas the 185 kDa protein is equally active on both substrates. The kinetic properties of the recombinant enzyme are similar to those reported for the native MTase derived from murine erythroleukemia cells. The new clones are capable of yielding large quantities of intact MTases for further structural and functional studies.     

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.     

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.     

Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme.

Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.     

Purification and in vitro complementation of mutant histidinol dehydrogenases.

The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface.     

Characterization of an<Emphasis Type="Italic">Eco</Emphasis>R 124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit,which results in a novel DNA specificity

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.     

Purification and characterization of rice DNA methyltransferase

Epigenetic modification is essential for normal development and plays important roles in gene regulation in higher plants. Multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. We had previously cloned and characterized DNA methyltransferase (DNA MTase) gene homologues (OsMET1) from rice. In this present study, determination of DNA MTase activity in different cellular compartments showed that DNA MTase was enriched in nuclei and the activity was remarkably increased during imbibing dry seeds. We had optimized the purification technique for DNA MTase enzyme from shoots of 10-day-old rice seedlings using the three successive chromatographic columns. The Econo-Pac Q, the Hitrap-Heparin and the Superdex-200 columns yielded a protein fraction of a specific activity of 29, 298 and 800 purification folds, compared to the original nuclear extract, respectively. The purified protein preferred hemi-methylated DNA substrate, suggesting the maintenance activity of methylation. The native rice DNA MTase was approximately 160–170 kDa and exhibited a broad pH optimum in the range of 7.6 and 8.0. The enzyme kinetics and inhibitory effects by methyl donor analogs, base analogs, cations, and cationic amines on rice DNA MTase were examined. Global cytosine methylation status of rice genome during development and in various tissue culture systems were monitored and the results suggested that the cytosine methylation level is not directly correlated with the DNA MTase activity. The purification and characterization of rice DNA MTase enzyme are expected to enhance our understanding of this enzyme function and their possible contributions in Gramineae plant development.     

Crystal structure of MboIIA methyltransferase

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.     

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.     

Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.     

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonuclease HinfI.

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of HinfI digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.     

Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis.

Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.     

Cloning and expression of a β-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme

Abstract A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA , was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 °C) optima were determined.     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase

Summary Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.