Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified virus with tumorigenic potential. Here, we cloned and expressed the DNA polymerase (Pol-8) of KSHV and its processivity factor (PF-8). Pol-8 bound specifically to PF-8 in vitro. Moreover, the DNA synthesis activity of Pol-8 was shown in vitro to be strongly dependent on PF-8. Addition of PF-8 to Pol-8 allowed efficient synthesis of fully extended DNA products corresponding to the full-length M13 template (7,249 nucleotides), whereas Pol-8 alone could incorporate only several nucleotides. The specificity of PF-8 and Pol-8 for each other was demonstrated by their inability to be functionally replaced by the DNA polymerases and processivity factors of herpes simplex virus 1 and human herpesvirus 6.     

Characterization of human herpesvirus 8 ORF59 protein (PF-8) and mapping of the processivity and viral DNA polymerase-interacting domains

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.     

Kaposi’s sarcoma-associated herpesvirus processivity factor-8 dimerizes in cytoplasm before being translocated to nucleus

The processivity factor-8 (PF-8) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication. PF-8 forms homodimers in solution and is observed as a dimer on the DNA. Here, we show that PF-8 dimerizes in cells and that amino acid residues 1-21 and residues 277-304 of PF-8 (396R) are required for dimerization in vivo. Importantly, we demonstrate that PF-8 dimerizes in the cytoplasm before being translocated to the nucleus. The significance of PF-8 cytoplasmic dimerization as a possible first step in the formation of a prereplication complex is discussed.     

The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

Kaposi''s sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi''s sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 Å. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8''s mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.Most if not all organisms with DNA genomes have mechanisms to ensure processive DNA synthesis. In bacteria, archaea, and eukaryotes, DNA polymerase subunits include a catalytic subunit and a processivity factor, often referred to as a “sliding clamp.” In these organisms, a clamp loader protein is required to assemble the processivity factor onto the DNA (27, 37). The bacterial sliding (beta) clamp is made up of homodimers of a subunit that comprises three structurally similar subdomains (26), whereas archaeal and eukaryotic proliferating cell nuclear antigen (PCNA) is composed of homotrimers that comprise two structurally similar subdomains (27, 37). For both of these clamps, the monomers assemble head-to-tail to form a closed homodimeric or homotrimeric ring, respectively, around the DNA. In these organisms, a clamp loader protein is required to efficiently load the clamp onto DNA, using an ATP-dependent process. Once loaded on DNA, the processivity factor is capable of binding directly to the DNA polymerase, conferring extended strand synthesis without falling off of the template (50).Herpesviruses encode their own DNA polymerases. However, unlike bacteria, archaea, and eukaryotes, herpesviruses do not encode clamp loaders to assemble their processivity factors onto the DNA. Yet, the accessory subunits of the herpesvirus DNA polymerases still associate with DNA with nanomolar affinity to enable long-chain DNA synthesis (9, 16, 23, 25, 29, 35, 44, 46, 53, 56). Human herpesviruses are divided into three classes, namely, the alpha-, beta-, and gammaherpesviruses, based on homologies found in their genomic organization as well as in protein sequences and function (45). Crystal structures have been determined for the processivity factor UL42 from the alphaherpesvirus herpes simplex virus type 1 (HSV-1) and for UL44 from the betaherpesvirus human cytomegalovirus (HCMV) (2, 3, 58). Despite having little if any sequence homology with processivity factors outside of their herpesvirus subfamily, these structures all share the “processivity fold” originally seen in the structure of the bacterial beta clamp (26). Interestingly, some of these processivity factors have a different quaternary structure. PCNA forms a head-to-tail trimeric ring (18, 27), HSV-1 UL42 is a monomer (10, 14, 16, 46, 58) equivalent to one-third of the PCNA complex, and HCMV UL44 is a head-to-head dimer in the form of a C-shaped clamp (2, 3, 9).Both HSV-1 UL42 and HCMV UL44 have a basic face that has been shown to be important for interacting with DNA (25, 35). In the case of dimeric HCMV UL44, the basic surface of each monomer faces inward, toward the center of the C clamp, and includes a basic loop, called the “gap loop,” that is thought to wrap around DNA (24). Recently the crystal structure of the bacterial beta clamp was determined in complex with DNA (15). In that structure, DNA was found to be located in the central pore of the clamp. Amino acid residues that interacted with DNA were in positions structurally homologous to those found on the positively charged faces of UL42 and UL44.UL42 and UL44 each also has a surface, facing away from the DNA binding face, that is important for interacting with the catalytic subunit of the viral DNA polymerase. Indeed, both of these proteins have been crystallized in complex with C-terminal peptides from their respective catalytic subunits, HSV-1 UL30 and HCMV UL54 (2, 58). Together with biochemical and mutational analyses, these crystal structures indicated that, although the details of the interaction are different, the catalytic subunit of the polymerase binds to a region including and in close proximity to a long loop that connects the N- and C-terminal subdomains, called the interdomain connector loop (32-34). The corresponding region of PCNA is also important for polymerase attachment and mediates the interactions of PCNA with many other cellular proteins (40). Both UL54 and UL30 were shown to attach to their respective subunits, UL44 and UL42, by way of their extreme C termini. The C-terminal residues responsible for this interaction correspond to amino acids that are not detectably conserved, either by sequence or by structure, among herpesvirus catalytic subunits. The HSV-1 UL30-UL42 interaction involves a groove to one side of the UL42 connector loop, with hydrophilic interactions being critical (58). The HCMV UL54-UL44 interaction involves a crevice near the UL44 connector loop, and hydrophobic interactions are crucial (2, 32, 33). Moreover, the HCMV UL44 crevice is on the opposite side of the connector loop with respect to the HSV-1 UL42 groove.Kaposi''s sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus, encodes a viral DNA polymerase catalytic subunit, Pol-8, and an accessory subunit, PF-8 (4, 7, 8, 29, 48, 57). PF-8 can bind to Pol-8 directly and specifically (8, 29) and is required for long-chain DNA synthesis in vitro (29). Similarly to UL44, PF-8 forms dimers in solution and when bound to DNA (9). Although it is likely that UL44 and PF-8 are the processivity factors for HCMV and KSHV, respectively, rigorous experiments demonstrating this have not been performed. However, for the sake of brevity and clarity, we will refer to these proteins as processivity factors.Here we present the crystal structure of PF-8 and show that PF-8 forms a head-to-head homodimer akin to UL44 but lacking the long gap loops which are thought to wrap around DNA. This suggests that PF-8 binds DNA differently than does UL44 or UL42. Because Pol-8 appears to lack a long, flexible C-terminal tail with a length comparable to those of other herpesvirus Pols, we expect the mode of binding of the catalytic subunit to be different as well. Based on structural data, information from homologs, and initial biochemical results, we were able to identify possible sites for interactions with DNA and Pol-8 and to propose a model for the simultaneous interaction of all three components of the complex. Further, the availability of crystal structures for all three herpesvirus classes provides new insights into comparative structure, function, and evolution.     

The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer

The human cytomegalovirus DNA polymerase consists of a catalytic subunit, UL54, and a presumed processivity factor, UL44. We have solved the crystal structure of residues 1-290 of UL44 to 1.85 A resolution by multiwavelength anomalous dispersion. The structure reveals a dimer of UL44 in the shape of a C clamp. Each monomer of UL44 shares its overall fold with other processivity factors, including herpes simplex virus UL42, which is a monomer that binds DNA directly, and the sliding clamp, PCNA, which is a trimer that surrounds DNA, although these proteins share no obvious sequence homology. Analytical ultracentrifugation and gel filtration measurements demonstrated that UL44 also forms a dimer in solution, and substitution of large hydrophobic residues along the homodimer interface with alanine disrupted dimerization and decreased DNA binding. UL44 represents a hybrid processivity factor as it binds DNA directly like UL42, but forms a C clamp that may surround DNA like PCNA.     

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.     

Human herpesvirus 8 encodes a homolog of interleukin-6.

Kaposi's sarcoma is a multifocal lesion that is reported to be greatly influenced by cytokines such as interleukin-6 (IL-6) and oncostatin M. DNA sequences of a novel human gammaherpesvirus, termed human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus, have been identified in all epidemiological forms of Kaposi's sarcoma with high frequency. The presence of HHV-8 DNA is also clearly associated with certain B-cell lymphomas (body cavity-based lymphomas) and multicentric Castleman's disease. Sequence analysis of a 17-kb fragment revealed that adjacent to a block of conserved herpesvirus genes (major DNA-binding protein, glycoprotein B, and DNA polymerase), the genome of HHV-8 encodes structural homolog of IL-6. This cytokine is involved not only in the pathogenesis of Kaposi's sarcoma but also in certain B-cell lymphomas and multicentric Castleman's disease. The viral counterpart of IL-6 (vIL-6) has conserved important features such as cysteine residues involved in disulfide bridging or an amino-terminal signal peptide. Most notably, the region known to be involved in receptor binding is highly conserved in vIL-6. This conservation of essential features and the remarkable overlap between diseases associated with HHV-8 and diseases associated with IL-6 disregulation clearly suggest that vIL-6 is involved in HHV-8 pathogenesis.     

Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver

Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 microM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication Clamps and Clamp Loaders

To achieve the high degree of processivity required for DNA replication, DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for replication. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.During each round of DNA replication, thousands to billions of nucleotides must be faithfully copied in a short period of time. However, by themselves, replicative DNA polymerases are distributive, synthesizing only ten or so nucleotides of complementary DNA before dissociating. To achieve the high degree of processivity required for efficient DNA replication, replicative DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. Such an association effectively tethers the polymerase to DNA, substantially increasing the amount of continuous replication. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for interaction with DNA polymerases. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.     

Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7

Bacteriophage T7 gene 5 protein has been purified to apparent homogeneity from cells overexpressing its gene several hundred-fold. Gene 5 protein is a DNA polymerase with low processivity; it dissociates from the primer-template after catalyzing the incorporation of 1-50 nucleotides, depending on the salt concentration. Escherichia coli thioredoxin, a host protein that is tightly associated with the gene 5 protein in phage-infected cells, is not required for this activity. Thioredoxin acts as an accessory protein to bestow processivity on the polymerizing reaction; DNA synthesis catalyzed by the gene 5 protein-thioredoxin complex on a single-stranded DNA template can polymerize thousands of nucleotides without dissociation. Conditions that increase the stability of secondary structures in the template (i.e., low temperature or high ionic strength) decrease the processivity. E. coli single-stranded DNA-binding protein stimulates both the rate of elongation and the processivity of the gene 5 protein-thioredoxin complex.     

Analysis of processivity of mungbean dideoxynucleotide-sensitive DNA polymerase and detection of the activity and expression of the enzyme in the meristematic and meiotic tissues and following DNA damaging agent

Analysis of the processivity of mungbean ddNTP-sensitive DNA polymerase showed the incorporation of ∼35-40 nucleotides per binding event in the replication assays involving M13 ss DNA template with 5′-labeled 17-mer primer. Optimal processivity was obtained with 100-150 mM KCl and 6-8 mM Mg²⁺ at pH 7.5. The enzyme showed preference for Mg²⁺ over Mn²⁺ as the metal activator for processivity. 2′, 3′ dideoxythymidine 5′ triphosphate (ddTTP) and rat DNA pol β antibody strongly influenced distributive synthesis. Considerable enhancement in processivity was noticed at 1 mM ATP and 2-4 mM spermidine while higher concentrations of spermidine caused distributive synthesis. The enzyme was found to be active in both meristematic and meiotic tissues and distinctly induced by EMS treatment. DNA-binding assays revealed distinct binding ability of the enzyme to template/primer and damaged DNA substrate. Together these observations illustrate the probable involvement of the enzyme in replication and repair machinery in higher plants.     

Processivity of the DNA polymerase alpha-primase complex from calf thymus

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.     

Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp

Y-family DNA polymerases can extend primer strands across template strand lesions that stall replicative polymerases. The poor processivity and fidelity of these enzymes, key to their biological role, requires that their access to the primer-template junction is both facilitated and regulated in order to minimize mutations. These features are believed to be provided by interaction with processivity factors, beta-clamp or proliferating cell nuclear antigen (PCNA), which are also essential for the function of replicative DNA polymerases. The basis for this interaction is revealed by the crystal structure of the complex between the 'little finger' domain of the Y-family DNA polymerase Pol IV and the beta-clamp processivity factor, both from Escherichia coli. The main interaction involves a C-terminal peptide of Pol IV, and is similar to interactions seen between isolated peptides and other processivity factors. However, this first structure of an entire domain of a binding partner with an assembled clamp reveals a substantial secondary interface, which maintains the polymerase in an inactive orientation, and may regulate the switch between replicative and Y-family DNA polymerases in response to a template strand lesion.     

The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2

v-cyclin encoded by Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV or HHV8) associates with cellular cyclin-dependent kinase 6 (CDK6) to form a kinase complex that promotes cell-cycle progression, but can also induce apoptosis in cells with high levels of CDK6. Here we show that whereas HHV8-encoded v-Bcl-2 protects against this apoptosis, cellular Bcl-2 has lost its anti-apoptotic potential as a result of an inactivating phosphorylation in its unstructured loop region. Moreover, we identify Bcl-2 as a new substrate for v-cyclin-CDK6 in vitro, and show that it is present in a complex with CDK6 in cell lysates. A Bcl-2 mutant with a S70A S87A double substitution in the loop region is not phosphorylated and provides resistance to apoptosis, indicating that inactivation of Bcl-2 by v-cyclin-CDK6 may be required for the observed apoptosis. Furthermore, the identification of phosphorylated Bcl-2 in HHV8-positive Kaposi's sarcoma indicates that HHV8-mediated interference with host apoptotic signalling pathways may encourage the development of Kaposi's sarcoma.     

Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.     

Spindle cells and their role in Kaposi's sarcoma

Spindle cells represent the main cell type of the advanced final nodular stage of Kaposi's sarcoma lesions. Despite some clinical and epidemiological differences, the four Kaposi's sarcoma forms (classic, endemic, post-transplant and epidemic) display very similar histopathological features, with the proliferation of spindle cells (considered as the Kaposi's sarcoma tumor cells) associated with inflammation and neo-angiogenesis. Electron-microscopy and immuno-histochemistry studies have led to the consensus that the spindle cells originated from the endothelial lineage. However, only recently, studies that used specific lymphatic immunological markers (such as podoplanin) and molecular features (gene expression microarrays) strongly linked Kaposi's sarcoma spindle cells to the endothelium lymphatic cell lineage. Both hybridization and immuno-histochemistry techniques have demonstrated that human herpesvirus 8 also known as Kaposi's sarcoma associated herpesvirus was present in spindle cells at all stages of the disease (patch, plaque, nodule). Interestingly, while the human herpesvirus 8 latent genes are expressed in nearly all tumor spindle cells, only a small fraction of them expresses markers of viral lytic replication. Recent findings showing that nodular Kaposi's sarcoma lesions display all patterns of human herpesvirus 8 clonality support the model according to which this tumor begins as a polyclonal disease with a subsequent evolution to a mono/oligoclonal process involving infected spindle cells. Spindle cells appear to be the central masterpiece in KS tumorigenesis, however the exact respective role of each human herpesvirus 8 gene, in the initiation and the disease progression is still under investigation and the question of whether or not this tumor is a reactive process or a true malignant proliferation of spindle cells remains yet unclear.     

A covalent linkage between the gene 5 DNA polymerase of bacteriophage T7 and Escherichia coli thioredoxin,the processivity factor: fate of thioredoxin during DNA synthesis

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.