The herpes simplex virus DNA polymerase: Analysis of the functional domains

The structural and functional organization of the herpes simplex virus type I (HSV-1) DNA polymerase enzyme of strain ANG was studied by a combination of sequence and immunobiochemical analyses. Comparison of the HSV-1 ANG DNA polymerase sequence with those of pro- and eukaryotic DNA polymerases resulted in the allocation of eleven conserved regions within the HSV-1 DNA polymerase. From the analysis of all currently identified mutations of temperature-sensitive and drug-resistant HSV-1 DNA polymerase mutants as well as from the degree of conservancy observed, it could be deduced that the amino-acid residues 597–961, comprising the homologous sequence regions IV–IX, constitute the major structural components of the catalytic domain of the enzyme which should accommodate the sites for polymerizing and 3′-to-5′ exonucleolytic functions. Further insight into the structural organization was gained by the use of polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the ANG polymerase. Each of the antisera was able to immunostain as well as to immunoprecipitate a viral polypeptide of 132 ± 5 kDa that corresponded well to the molecular mass of 136 kDa predicted from the coding sequences. Enzyme-binding and neutralization studies confirmed that both functions, polymerase and 3′-to-5′ exonuclease, are intimately related to each other, and revealed that, in addition to the sequences of the proposed catalytic domain, the very C-terminal sequences, except for amino-acid residues 1072–1146, are important for the catalytic functions of the enzyme, most likely effecting the binding to DNA.     

Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly

Pierisin-1, a cytotoxic protein found naturally in the cabbage butterfly, induces apoptosis of mammalian cells. Our recent studies suggest that pierisin-1 consists of an N-terminal ADP-ribosyltransferase domain, and a C-terminal region that binds to receptors on the surfaces of target cells and incorporates the protein into cells. The present study was undertaken to identify receptors for pierisin-1. The cross-linking and cloning experiments suggested that the proteins on cell membrane had no binding ability to pierisin-1. Inhibitory assays of fractionated lipids from human cervical carcinoma HeLa cells, which are highly sensitive to pierisin-1, indicated neutral glycosphingolipids on the cell surface to show receptor activity. Inhibitory assays and TLC immunostaining using anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids as active components. Analysis of their structures with glycosphingolipid-specific antibodies and negative secondary ion mass spectrometry identified them as globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4 for pierisin-1 were also confirmed with these authentic compounds. Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1 receptors. The C-terminal region of pierisin-1 consists of possible lectin domains of a ricin B-chain, containing QXW sequences, which are essential for its structural organization. Alteration of QXW by site-directed mutagenesis caused marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal region, in a manner similar to ricin, and then exhibits cytotoxicity after incorporation into the cell.     

The ADP-ribosylating mosquitocidal toxin from Bacillus sphaericus: proteolytic activation, enzyme activity, and cytotoxic effects.

The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a approximately 97-kDa protein sharing sequence homology within the N terminus with the catalytic domains of various bacterial ADP-ribosyltransferases. Here we studied the proteolytic activation of the ADP-ribosyltransferase activity of MTX. Chymotrypsin treatment of the 97-kDa MTX holotoxin (MTX(30-870)) results in a 70-kDa putative binding component (MTX(265-870)) and a 27-kDa enzyme component (MTX(30-264)), possessing ADP-ribosyltransferase activity. Chymotryptic cleavage of an N-terminal 32-kDa fragment of MTX (MTX(30-308)) also yields MTX(30-264), but the resulting ADP-ribosyltransferase activity is much greater than that of the processed MTX(30-870). Kinetic studies revealed a K(m) NAD value of 45 microm for the processed 32-kDa MTX fragment, and a K(m) NAD value of 1300 microm for the processed holotoxin. Moreover, the k(cat) value for the activated MTX(30-308) fragment was about 10-fold higher than that for the activated holotoxin (MTX(30-870)). Precipitation analysis showed that the 70-kDa proteolytic fragment of MTX remains noncovalently bound to the N-terminal 27-kDa fragment, thereby inhibiting ADP-ribosyltransferase and NAD glycohydrolase activities. Glu(197) of MTX(30-264) was identified as the "catalytic" glutamate that is conserved in all ADP-ribosyltransferases. Whereas mutated MTX(30-264)E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activity, mutated MTX(30-264)E195Q possesses glycohydrolase activity but not transferase activity. Transfection of HeLa cells with a vector encoding a fusion protein of MTX(30-264) with a green fluorescent protein led to cytotoxic effects characterized by cell rounding and formation of filopodia-like protrusions. These cytotoxic effects were not observed with the catalytically inactive MTX(30-264)E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.     

Baculoviral expression of correctly processed ADAMTS proteins fused with the human IgG-Fc region

A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) is a novel family of extracellular proteases supposedly involved in inflammation, angiogenesis, development and coagulation. To overexpress the active ADAMTS proteins, we designed a chimeric molecule composed of a catalytic domain of ADAMTS-1 or -4 and the human IgG Fc region in a baculoviral expression system. Both ADAMTS-Fc fusions were produced efficiently in the baculovirus-infected insect cells. The purified fusions underwent cleavage at the predicted furin recognition site. Both ADAMTS-Fc fusions bound to alpha(2)-macroglobulin, further indicating that they were correctly processed with the catalytic activity in this system; however, they failed to digest the peptides derived from the aggrecan sequences known to be clipped by the native enzyme, possibly due to the lack of required multiple interactions existing between the native protease and physiological substrate. In conclusion, the high productivity and facilitated purification of the fusion proteins would offer the source of the biochemical, biophysical or structural studies on the catalytic domain of the ADAMTS proteins.     

Crystal structures of the type III effector protein AvrPphF and its chaperone reveal residues required for plant pathogenesis

The avrPphF locus from Pseudomonas syringae pv. phaseolicola, the causative agent of bean halo-blight disease, encodes proteins which either enhance virulence on susceptible hosts or elicit defense responses on hosts carrying the R1 resistance gene. Here we present the crystal structures of the two proteins from the avrPphF operon. The structure of AvrPphF ORF1 is strikingly reminiscent of type III chaperones from bacterial pathogens of animals, indicating structural conservation of these specialized chaperones, despite high sequence divergence. The AvrPphF ORF2 effector adopts a novel "mushroom"-like structure containing "head" and "stalk" subdomains. The head subdomain possesses limited structural homology to the catalytic domain of bacterial ADP-ribosyltransferases (ADP-RTs), though no ADP-RT activity was detected for AvrPphF ORF2 in standard assays. Nonetheless, this structural similarity identified two clusters of conserved surface-exposed residues important for both virulence mediated by AvrPphF ORF2 and recognition of this effector by bean plants expressing the R1 resistance gene.     

Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain.

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.     

ADP-ribosyltransferase is highly conserved: purification and characterization of ADP-ribosyltransferase from a fish and its comparison with the human enzyme

Summary Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11700-fold purified activity shows a major protein band at a molecular mass of 75000 kDa in a SDS-polyacrylamide gel.In situ reactivation of SDS gels showed the 75000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115000, 90000 and 87000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparentK _m for NAD⁺ of 24 M. The temperature optimum is 10°C compared with 25°C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout. ribosyl-)residues from NAD⁺. It plays a central role in processes affecting DNA function such as DNA repair, recombination, differentiation, tumorigenic cell transformation and cell proliferation (Pekala and Moss 1983; Ueda and Hayaishi 1985; Creissen and Shall 1982; Borek et al. 1984; Lunec 1984; Cleaver et al. 1985; Tseng et al. 1987). It seems to be a central controller of cell physiology (Loetscher et al. 1987; Schweiger et al. 1987). ADP-ribosylation is high in proliferating cells whereas it is low in differentiated cells (Surowy and Berger 1983). Due to the important functions of ADP-ribosyltransferase this enzyme has been studied extensively but almost exclusively in mammals (Ueda and Hayaishi 1985). Only little is known about ADP-ribosyltransferases of lower vertebrates and of species of lower phylogenetic families. Hence it appeared of interest to us to study this enzyme in a fish. Here we report the purification to homogeneity and the characterization of ADP-ribosyltransferase from trout liver. We compared the fish enzyme with the human one and found very similar enzymatic properties and immunological crossreactivity. This indicates that ADP-ribosyltransferases are highly conserved.     

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.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.     

Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli.

The RecB subunit of the Escherichia coli RecBCD enzyme has been shown in previous work to have two domains: an N-terminal 100 kDa domain with ATP-dependent helicase activity, and a C-terminal 30 kDa domain. The 30 kDa domain had nuclease activity when linked to a heterologous DNA binding protein, but by itself it appeared unable to bind DNA and lacked detectable nuclease activity. We have expressed and isolated this 30 kDa domain, called RecB(N), and show that it does have nuclease activity detectable at high protein concentration in the presence of polyethylene glycol, added as a molecular crowding agent. The activity is undetectable in a mutant RecB(N)protein in which an aspartate residue has been changed to alanine. Structural analysis of the wild-type and mutant RecB(N)proteins by second derivative absorbance and circular dichroism spectroscopy indicates that both are folded proteins with very similar secondary and tertiary structures. The results show that the Asp-->Ala mutation has not caused a significant structural change in the isolated domain and they support the conclusion that the C-terminal domain of RecB has the sole nuclease active site of RecBCD.     

Structural domains in the type III restriction endonuclease EcoP15I: characterization by limited proteolysis, mass spectrometry and insertional mutagenesis

The Type III restriction endonuclease EcoP15I forms a hetero-oligomeric enzyme complex that consists of two modification (Mod) subunits and two restriction (Res) subunits. Structural data on Type III restriction enzymes in general are lacking because of their remarkable size of more than 400 kDa and the laborious and low-yield protein purification procedures. We took advantage of the EcoP15I-overexpressing vector pQEP15 and affinity chromatography to generate a quantity of EcoP15I high enough for comprehensive proteolytic digestion studies and analyses of the proteolytic fragments by mass spectrometry. We show here that in the presence of specific DNA the entire Mod subunit is protected from trypsin digestion, whereas in the absence of DNA stable protein domains of the Mod subunit were not detected. In contrast, the Res subunit is comprised of two trypsin-resistant domains of approximately 77-79 kDa and 27-29 kDa, respectively. The cofactor ATP and the presence of DNA, either specific or unspecific, are important stabilizers of the Res subunit. The large N-terminal domain of Res contains numerous functional motifs that are predicted to be involved in ATP-binding and hydrolysis and/or DNA translocation. The C-terminal small domain harbours the catalytic center. Based on our data, we conclude that both structural Res domains are connected by a flexible linker region that spans 23 amino acid residues. To confirm this conclusion, we have investigated several EcoP15I enzyme mutants obtained by insertion mutagenesis in and around the predicted linker region within the Res subunit. All mutants tolerated the genetic manipulation and did not display loss of function or alteration of the DNA cleavage position.     

A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc

Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.     

Structure and mechanism of metallocarboxypeptidases

Metallocarboxpeptidases cleave C-terminal residues from peptide substrates and participate in a wide range of physiological processes, but they also contribute to human pathology. On the basis of structural information, we can distinguish between two groups of such metallopeptidases: cowrins and funnelins. Cowrins comprise protozoan, prokaryotic, and mammalian enzymes related to both neurolysin and angiotensin-converting enzyme and their catalytic domains contain 500-700 residues. They are ellipsoidal and traversed horizontally by a long, deep, narrow active-site cleft, in which the C-terminal residues are cut from oligopeptides and unstructured protein tails. The consensus cowrin structure contains a common core of 17 helices and a three-stranded beta-sheet, which participates in substrate binding. This protease family is characterized by a set of spatially conserved amino acids involved in catalysis, HEXXH+EXXS/G+H+Y/R+Y. Funnelins comprise structural relatives of the archetypal bovine carboxypeptidase A1 and feature mammalian, insect and bacterial proteins with strict carboxypeptidase activity. Their approximately 300-residue catalytic domains evince a consensus central eight-stranded beta-sheet flanked on either side by a total of eight helices. They also contain a characteristic set of conserved residues, HXXE+R+NR+H+Y+E, and their active-site clefts are rather shallow and lie at the bottom of a funnel-like cavity. Therefore, these enzymes act on a large variety of well-folded proteins. In both cowrins and funnelins, substrate hydrolysis follows a common general base/acid mechanism. A metal-bound solvent molecule ultimately performs the attack on the scissile peptide bond with the assistance of a strictly conserved glutamate residue.     

Membrane enzyme systems responsible for the Ca(2+)-dependent phosphorylation of Ser(27), the independent phosphorylation of Tyr(10) and Tyr(7), and the dephosphorylation of these phosphorylated residues in the alpha-chain of H/K-ATPase

H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) residues of the alpha-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca(2)(+) (K(0.5) = 0.9 microM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCalpha and PKCbetaII. The addition of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of alpha-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G1 membrane and others indicate the apparent molecular weight of the Src-kinase to be approximately 60 kDa, the PKCalpha and/or PKCbII to be approximately 80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be approximately 35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.     

The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.

DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.     

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.