Characterization of human factor VIII and interaction with von Willebrand factor. An electron microscopic study

Blood coagulation factor VIII is a large glycoprotein that circulates in plasma at relative low concentration (0.1 microgram/ml). It consists of a heterogeneous mixture of a series heavy-chain peptides (90-200 kDa), each associated with a light chain of 80 kDa. To gain insight into the physical properties of the protein, we have characterized purified human factor VIII by electron microscopy and rotary shadowing. Electron microscopy of rotary shadowed factor VIII molecules showed predominantly a single globular domain structure, with a somewhat asymmetric shape, while two-domain structures were also encountered. The overall dimensions of the globular domains ranged from 4 x 6 nm to 8 x 12 nm. EDTA treatment of factor VIII reduced the overall dimensions (2.5 x 5 nm to 6 x 10 nm) while treatment with thrombin reduced the dimensions to a small extent. In complexes with von Willebrand factor, factor VIII appeared localized at the globular domains of von Willebrand factor multimers. In addition, incubation of factor VIII with Staphylococcus aureus V8 protease fragments SpII and SpIII revealed only binding to the globular domains of SpIII. In this study, the first morphological characterization of human factor VIII is presented, together with its direct localization on von Willebrand factor multimers.     

Purification and characterization of mammalian DNA methyltransferases by use of monoclonal antibodies

Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.     

Distinct CG and CNG DNA methyltransferases in Pisum sativum

DNA methyltransferase activity, present in low salt extracts of nuclei from young pea shoot apices, has been fractionated into two different species by assaying with model substrates. The CG methyltransferase (an unstable enzyme believed to be of 140 kDa) methylates cytosine only in oligonucleotides with CG and CI dinucleotide targets while an enzyme of 110 kDa (the CNG methyltransferase) methylates the cytosines in 5′-CAG-3′ and 5′-CTG-3′ target sequences, especially when hemimethylated, but not in 5′-CCG-3′ nor in 5′-CGG-3′ target sequences present in oligonucleotides.     

Structural model of factors V and Va based on scanning transmission electron microscope images and mass analysis

Coagulation factor V (fV) is a single-chain glycoprotein (Mr 330,000; domain structure A1-A2-B-A3-C1-C2) that is activated to factor Va (fVa; Mr 174,000) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2; Mr 94,000) and a light chain (A3-C1-C2; Mr 74,000). We analyzed the ultrastructure of scanning transmission electron microscope images of bovine and human fV, bovine fVa, and its constituent light chains and heavy chains. Factor V molecules had irregularly globular (10-12 nm) to oblong (8-14 nm) core structures which commonly displayed a peripheral satellite appendage of variable morphology attached to the core by a narrow stalk. Scanning transmission electron microscope mass analyses indicated that monomolecular bovine fV molecules had a mass of 322 +/- 45 kDa and human fV, 315 +/- 31 kDa. Factor Va molecules were irregular, globular (8-12 nm) structures that resembled the fV core structure, lacked the satellite appendage representing B domainal structures, and had a mass of 180 +/- 22 kDa. Our findings permit us to propose a structural model of fV suggesting the relative orientation of its closely associated light chain and heavy chain core components and indicating that these constituents remain associated in the transition from fV to fVa.     

Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.     

De novo and maintenance DNA methylation by a mouse plasmacytoma cell DNA methyltransferase

A DNA methyltransferase of Mr = 140,000 that is active on both unmethylated and hemimethylated DNA substrates has been purified from the murine plasma-cytoma cell line MPC 11. The maximal rate of methylation was obtained with maintenance methylation of hemimethylated Micrococcus luteus or M13 DNAs. At low enzyme concentrations, the highest rate of de novo methylation occurred with single-stranded DNA or relatively short duplex DNA containing single-stranded regions. Strong substrate inhibition was observed with hemimethylated but not unmethylated DNA substrates. Fully methylated single-stranded M13 phage DNA inhibited neither the de novo nor the maintenance reactions, but unmethylated single-stranded M13 DNA strongly inhibited the maintenance reaction. The kinetics observed with hemimethylated and single-stranded substrates could be explained if the enzyme were to bind irreversibly to a DNA molecule and to aggregate if present in molar excess. Such aggregates would be required for activity upon hemimethylated but not single-stranded DNA. For de novo methylation of duplex DNA, single-stranded regions or large amounts of methyltransferase appear to be required. The relative substrate preference for the enzyme is hemimethylated DNA greater than fully or partially single-stranded DNA greater than fully duplex DNA.     

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.     

Purification and properties of a novel DNA methyltransferase from cultured rice cells

DNA methyltransferase activity has been observed in a total crude homogenate of rice cells grown in suspension culture using either native plant DNA or, under the conditions used, the more responsive hemimethylated poly (dI-MedC).poly(dI-dC). Using the latter substrate we have purified an enzyme fraction 380-fold by salt extraction of chromatin, DEAE cellulose and phosphocellulose. This purified fraction showed enzyme activity only with poly (dI-MedC).poly(dI-dC) thus suggesting the occurrence in plants of a DNA methyltransferase specific for hemimethylated DNA. A Mr value of 54000 was calculated on the basis of the sedimentation coefficient which was determined by sucrose density gradient centrifugation. Apparent Km values for poly (dI-MedC).poly(dI-dC) and S-adenosyl-L-methionine were found to be 17 micrograms/ml and 2.6 microM, respectively.     

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.     

Properties of a purified nuclear topoisomerase from L1210 cells

A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.     

The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.     

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites

We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.     

Crystalline green 5-hydroxyvaleryl-CoA dehydratase from Clostridium aminovalericum

A green enzyme from Clostridium aminovalericum with valeryl-CoA dehydrogenase activity was purified to homogeneity (169 +/- 3 kDa) and crystallized. By SDS/PAGE, one type of subunit (42 kDa) was detected indicating a homotetrameric structure. The unusual ultraviolet/visible spectrum of the green enzyme (maxima at 394 nm, 438 nm and 715 nm) was converted to a normal flavoprotein spectrum either by reduction with dithionite and reoxidation under air, or by removal of the prosthetic group at pH 2 and reconstitution with FAD (not FMN). Besides FAD (4 mol/169 kDa), the enzyme contained 4 mol of a CoA ester which was similar but not identical to 5-hydroxy-2-pentenoyl-CoA. The reconstituted holoenzyme as well as the native green enzyme, but not the apoenzyme, catalysed the reversible dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA in the absence of an external electron acceptor. In its presence (preferentially ferricenium ion), the green or yellow enzyme catalysed the formation of (E)-5-hydroxy-2-pentenoyl-CoA and 2,4-pentadienoyl-CoA either from 4-pentenoyl-CoA or from 5-hydroxyvaleryl-CoA. The reversible hydration of 2,4-pentadienoyl-CoA to (E)-5-hydroxy-2-pentenoyl-CoA was mediated by both enzymes as well as by the apoenzyme in the absence of FAD. Hydration of 4-pentenoate in 2H2O yielded optically active 5-hydroxy[2,4-2H2]valerate by the combined action of 5-hydroxyvalerate CoA-transferase, the green dehydratase and catalytical amounts of acetyl-CoA. The data show that the reversible hydration of the isolated double bond of 4-pentenoyl-CoA to 5-hydroxyvaleryl-CoA. which apparently violates the Markovnikov rule, is preceded by oxidation to 2,4-pentadienoyl-CoA. The latter compound, a vinyl analogue of 2-enoyl-CoA, is then easily hydrated to (E)-5-hydroxy-2-pentenoyl-CoA and finally reduced to 5-hydroxyvaleryl-CoA.     

Mechanism of action of eukaryotic DNA methyltransferase: Use of 5-azacytosine-containing DNA

Hemimethylated DNA substrates prepared from cell cultures treated with 5-azacytidine are efficient acceptors of methyl groups from S-adenosylmethionine in the presence of a crude preparation of mouse spleen DNA methyltransferase. Partially purified methyltransferase was also capable of efficiently modifying single-stranded unmethylated DNA. The methylation of single-stranded DNA was less sensitive to inhibition by salt than duplex DNA. The presence of other DNA species in the reaction mix (duplex or single-stranded, methylated or unmethylated) inhibited the modification of the hemimethylated duplex DNA. The enzyme was specific for DNA, since the presence of RNA in reaction mixtures did not inhibit the methylation of DNA. DNA methyltransferase formed a tight-binding complex with hemimethylated duplex DNA containing high levels of 5-azacytosine, and this complex was not dissociated by high concentrations of salt. Treatment of cultured cells with biologically effective concentrations of 5-azacytidine and other cytidine analogs modified in the 5 position resulted in a loss of extractable active enzyme from the cells. The amount of extractable active enzyme recovered slowly with time after treatment. These results suggest that incorporation of 5-azacytidine into DNA inhibits the progress of DNA methyltransferase along the duplex, perhaps by the formation of a tight-binding complex. This complex formation might be irreversible, so that new enzyme synthesis might be required to reverse the block of DNA methylation.     

Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy

The structure of the multisubunit yeast DNA polymerase epsilon (Pol epsilon) was determined to 20-A resolution using cryo-EM and single-particle image analysis. A globular domain comprising the catalytic Pol2 subunit is flexibly connected to an extended structure formed by subunits Dpb2, Dpb3 and Dpb4. Consistent with the reported involvement of the latter in interaction with nucleic acids, the Dpb portion of the structure directly faces a single cleft in the Pol2 subunit that seems wide enough to accommodate double-stranded DNA. Primer-extension experiments reveal that Pol epsilon processivity requires a minimum length of primer-template duplex that corresponds to the dimensions of the extended Dpb structure. Together, these observations suggest a mechanism for interaction of Pol epsilon with DNA that might explain how the structure of the enzyme contributes to its intrinsic processivity.     

Structure of initiation factor eIF-3 from rat liver. Hydrodynamic and electron microscopic investigations

On the basis of hydrodynamic, electron microscopic and biochemical investigations a new model of the structure of initiation factor eIF-3 is proposed. From sedimentation and diffusion coefficients of 16.35 S and 2.13 X 10(-7) cm2/s, respectively, as well as from sedimentation equilibrium measurements, a molecular mass of about 650 kDa was determined for isolated eIF-3. This is in agreement with molecular mass estimations by sodium dodecyl sulphate gel electrophoresis. A partial specific volume of 0.723 cm3/g was determined by means of the amino acid composition and the specific volume increments of the amino acids. From this value and from the molecular mass, a volume of 780 nm3 was calculated for eIF-3. In electron micrographs of isolated eIF-3, images with triangular profiles and side lengths of 14 nm, 16 nm, and 17 nm have been observed. Taking into account the calculated volume and considering the triangular image as one face of the particle, it is suggested that eIF-3 has the shape of a flat triangular prism with a height of about 7 nm and the above-mentioned side-lengths. This model is in agreement with results of electron microscopic investigations of eIF-3 in native small ribosomal subunits [Lutsch, G., Benndorf, R., Westermann, P., Bommer, U.-A. & Bielka, H. (1986) Eur. J. Cell Biol. 40/2, in press]. The high frictional ratio of about 1.7 also supports eIF-3 to be rather of a flat than of a globular shape.     

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.     

Molecular characterization of the human platelet integrin GPIIb/IIIa and its constituent glycoproteins

Human platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer, the integrin GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Below the critical micellar concentration of Triton X100 (TtX), the three glycoproteins do not bind appreciably to TtX and form association products of large size. The size-exclusion chromatographic patterns of GPIIb, GPIIIa and GPIIb/IIIa have been obtained at 0.2% TtX, and the molecular properties of the association products and monomer fractions have been determined by analysis of the detergent bound to the glycoproteins, laser-light scattering, sedimentation velocity, and electron microscopy (TEM). The monomer of the GPIIb-TtX complex was identified by the molecular mass (M) of the glycoprotein moiety (125 +/- 15 kDa), the molecular size (9.5 +/- 1.5 nm x 11 +/- 1.5 nm) and globular shape observed by TEM. It has a molecular mass (M*) of 197 +/- 20 kDa, a sedimentation coefficient s degrees 20* of 5.8 +/- 0.1 S, a Stokes radius R s* of 6.8 +/- 0.4 nm, and a frictional ratio f*/fmin* of 1.7 +/- 0.14. The (GPIIb)n-TtX complexes are disulphide-bonded size-heterogeneous association products of GPIIb, tetramers being the smallest species found. GPIIIa has a greater propensity to self-associate than GPIIb, this tendency being lower below 1 mg GPIIIa/ml, 0.1 mM Ca2+, pH 9.0. The (GPIIIa)n-TtX complexes are noncovalent size-heterogeneous association products of GPIIIa, tetramers being the smallest form observed. The monomer of the GPIIIa-TtX complex was identified by the 103 +/- 15 kDa M determined for the glycoprotein moiety, and the 9 +/- 1.5 nm x 10 +/- 1.5 nm size and globular shape observed by TEM. It has a M* of 136 +/- 15 kDa, a s degrees 20* of 3.9 +/- 0.3 S, a Rs* of 6.4 +/- 0.5 nm, a f*/fmin* of 1.9 +/- 0.3, and, when stored at pH 7.4, has a certain tendency to form filamentous association products (20-70 nm x 2-5 nm), as observed by TEM. The GPIIb/IIIa-TtX complex in 0.2% TtX/0.1 mM Ca2+ elutes as a single monomeric fraction, as deduced from the 210 +/- 15 kDa M determined for its glycoprotein moiety and the 12 +/- 1.5 nm x 14 +/- 1.5 nm size of the globular forms observed by TEM.(ABSTRACT TRUNCATED AT 400 WORDS)