In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Polymerase assays for lead discovery: An overall review of methodologies and approaches.

Polymerases represent an attractive molecular target for antibacterial drug development, antiviral intervention and cancer therapy. Over the past decade, academic groups and scientists from pharmaceutical industry have developed a large plethora of different functional assays to monitor the enzymatic reaction catalyzed by polymerases. These assays were used to enable high-throughput screening (HTS) for lead discovery purposes, as well as hit-to-lead (H2L) drug profiling activities. In both cases the choice of the assay technology is critical and to the best of our knowledge, there is no review available to help scientists to choose the most suitable assay. This review summarizes the most common functional assays developed to monitor the enzymatic activity of polymerases and discusses the advantages and disadvantages of each assay. Assays are presented and evaluated in term of cost, ease of use, high-throughput screening compatibility and liability towards delivering false positives and false negatives.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Comparison of two laboratory extraction techniques for the detection of Epstein-Barr virus in the saliva of nasopharyngeal carcinoma patients.

AIM: The aim of the present study was to compare the effectiveness of DNA extraction using an extraction kit against the standard boiling technique for the detection of Epstein-Barr virus (EBV) DNA in nasopharyngeal carcinoma (NPC) patients. METHODS: Stimulated whole saliva samples from newly-diagnosed NPC patients were collected. EBV DNA was extracted by both techniques (n = 23) followed by quantitative real-time polymerase chain reaction (PCR) using the primer/probe set for BALF5. RESULTS: The results of the quantitative real-time PCR were reproducible in both groups. The two techniques were moderately correlated (r = 0.67, P < 0.05), and the degree of agreement was good. However, the mean EBV DNA level in the boiling group (3.02 ± 8.67 × 10(6) copies/µL) was significantly higher than the extraction kit group (1.15 ± 2.66 × 10(6) copies/µL) (P < 0.05). The EBV DNA level was higher in patients at an advanced overall stage (P = 0.05). CONCLUSION: The results of the present study showed that the performance of the extraction kit was not superior to the simple boiling technique for the detection of salivary EBV DNA in NPC patients using real-time PCR. The salivary EBV DNA level in patients at an advanced overall stage appeared to be higher than in patients at an early stage.

Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation.

Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.

Isolation of a novel adenovirus from Rousettus leschenaultii bats from India.

Surveillance work was initiated to study the presence of highly infectious diseases like Ebola-Reston, Marburg, Nipah and other possible viruses that are known to be found in the bat species and responsible for causing diseases in humans. A novel adenovirus was isolated from a common species of fruit bat (Rousettus leschenaultii) captured in Maharashtra State, India. Partial sequence analysis of the DNA polymerase gene shows this isolate to be a newly recognized member of the genus Mastadenovirus (family Adenoviridae), approximately 20% divergent at the nucleotide level from Japanese BatAdV, its closest known relative.

Investigation of ornamental fish entering the EU for the presence of ranaviruses.

A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.

DNA damage induced Pol eta recruitment takes place independently of the cell cycle phase.

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol eta is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G(1). This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol eta recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol eta to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol eta to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.

Novel evidences for a tumor suppressor role of Rev3, the catalytic subunit of Pol zeta.

Cell cycle checkpoints and DNA repair act in concert to ensure DNA integrity during perturbation of normal replication or in response to genotoxic agents. Deficiencies in these protective mechanisms can lead to cellular transformation and ultimately tumorigenesis. Here we focused on Rev3, the catalytic subunit of the low-fidelity DNA repair polymerase zeta. Rev3 is believed to play a role in double-strand break (DSB)-induced DNA repair by homologous recombination. In line with this hypothesis, we show the accumulation of chromatin-bound Rev3 protein in late S-G2 of untreated cells and in response to clastogenic DNA damage as well as an gamma-H2AX accumulation in Rev3-depleted cells. Moreover, serine 995 of Rev3 is in vitro phosphorylated by the DSB-inducible checkpoint kinase, Chk2. Our data also disclose a significant reduction of rev3 gene expression in 74 colon carcinomas when compared to the normal adjacent tissues. This reduced expression is independent of the carcinoma stages, suggesting that the downregulation of rev3 might have occurred early during tumorigenesis.

The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm.

BACKGROUND: Human mitochondrial DNA (mtDNA) encodes 13 polypeptides of the electron transfer chain. Its replication is dependent on the nuclear-encoded polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM). For POLG, only the polyglutamine tract, characterized by a series of CAG repeats, has been investigated in human sperm. However, TFAM is associated with the reduction in mtDNA content of testicular sperm. We have determined whether POLG and TFAM have functional roles in post-ejaculatory sperm mtDNA. METHODS: Sperm samples were categorized as: normals, samples with one or two abnormal sperm parameters and oligoasthenoteratozoospermics (OATs). These were analysed by fluorescent PCR to determine the number of CAG repeats, real-time PCR for mtDNA copy number and immunocytochemistry and western blotting for patterns of expression for POLG, TFAM and the mtDNA-encoded COXI. RESULTS: Only the OAT group presented with a significantly higher incidence of heterozygosity for CAG repeats, higher mtDNA content and a lower percentage of sperm expressing POLG and TFAM. Paradoxically, good-quality sperm had fewer mtDNA copies but significantly more sperm expressed POLG, TFAM and COXI. CONCLUSIONS: Our data support the original findings that an association between sperm quality and POLG CAG repeats does exist. However, the biological significance of these variants in male infertility remains unclear, as these do not seem to affect mtDNA maintenance. The reduction in mtDNA content in normal samples likely reflects normal spermiogenesis, whereas increases in POLG and TFAM expression possibly compensate for the low mtDNA content, maintaining mitochondrial homeostasis.

Comparison of the Digene Hybrid Capture System Cytomegalovirus (CMV) DNA (version 2.0), Roche CMV UL54 analyte-specific reagent, and QIAGEN RealArt CMV LightCycler PCR reagent tests using AcroMetrix OptiQuant CMV DNA quantification panels and specimens from allogeneic-stem-cell transplant recipients.

The Digene Hybrid Capture system cytomegalovirus (CMV) DNA (version 2.0), Roche CMV UL54 analyte-specific reagent, and QIAGEN RealArt CMV LightCycler PCR reagent tests were compared using whole-virus standards and plasma specimens collected from allogeneic-stem-cell transplant recipients. PCR assays showed better speed, sensitivity, and specificity.

A novel role of DNA polymerase eta in modulating cellular sensitivity to chemotherapeutic agents.

Genetic defects in polymerase eta (pol eta; hRad30a gene) result in xeroderma pigmentosum variant syndrome (XP-V), and XP-V patients are sensitive to sunlight and highly prone to cancer development. Here, we show that pol eta plays a significant role in modulating cellular sensitivity to DNA-targeting anticancer agents. When compared with normal human fibroblast cells, pol eta-deficient cells derived from XP-V patients were 3-fold more sensitive to beta-d-arabinofuranosylcytosine, gemcitabine, or cis-diamminedichloroplatinum (cisplatin) single-agent treatments and at least 10-fold more sensitive to the gemcitabine/cisplatin combination treatment, a commonly used clinical regimen for treating a wide spectrum of cancers. Cellular and biochemical analyses strongly suggested that the higher sensitivity of XP-V cells to these agents was due to the inability of pol eta-deficient cells to help resume the DNA replication process paused by the gemcitabine/cisplatin-introduced DNA lesions. These results indicated that pol eta can play an important role in determining the cellular sensitivity to therapeutic agents. The findings not only illuminate pol eta as a potential pharmacologic target for developing new anticancer agents but also provide new directions for improving future chemotherapy regimen design considering the use of nucleoside analogues and cisplatin derivatives.

Structural analysis of the human neuroblastoma DNA replication complex: insights into faulty proliferation.

BACKGROUND: Whether faulty DNA synthesis contributes to neuroblastoma (NB) pathogenesis has received little attention. We have shown that NB DNA replication is orchestrated by a multiprotein DNA replication complex (DNA synthesome) which mediates error-prone DNA synthesis. We sought to define proteomic alterations of the NB DNA synthesome, which lead to lowered replication fidelity. METHODS: DNA synthesomes from NB and neural stem (NS) cell culture were purified. Proteomic differences of the DNA synthesome between NB and NS cells were determined using 2-dimensional polyacrylamide gel electrophoresis and immunodetection. RESULTS: DNA synthesome proteins from NB and NS cells were compared. Only replication protein A (RPA) showed distinct changes. RPA from NS cells resolved as a single spot (isoelectric point [pI] 5.75), whereas RPA in NB showed a main spot (pI 5.75) along with 3 additional isoforms. RPA binds to single-stranded DNA and participates in protein-protein interactions. CONCLUSIONS: NB DNA synthesome showed 3 distinct isoforms of RPA when compared with NS cells. These findings are significant in that it is possible to link changes in the fidelity of DNA replication with a specific protein alteration of the NB DNA synthetic apparatus. The novel RPA forms may be a new signature of NB malignancy.

Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

PURPOSE: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. METHODS: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. RESULTS: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. CONCLUSIONS: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

Identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase.

We report here on the identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase. Immunocytochemical experiments confirm that the enzyme indeed localizes to mitochondrial compartment. Inhibition of expression of the enzyme by RNA interference results in significant shortening of the poly(A) tails of the mitochondrial ND3, COX III and ATP 6/8 transcripts, suggesting that the investigated protein represents a bona fide mitochondrial poly(A) polymerase. This is in agreement with our sequencing data which show that poly(A) tails of several mitochondrial messengers are composed almost exclusively of adenosine residues. Moreover, the data presented here indicate that all analyzed mitochondrial transcripts with profoundly shortened poly(A) tails are relatively stable, which in turn argues against the direct role of long poly(A) extensions in the stabilization of human mitochondrial messengers.

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells.

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.

The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant.

To determine the possible role of polymerase eta (pol eta) in somatic hypermutation of B cells, a mutational analysis of 24 nonproductive rearrangements from a patient with xeroderma pigmentosum variant with a defect in pol eta was conducted. Although the mutational frequency of A and T bases decreased in WA (A/T, A) motifs, regardless of their RGYW (purine, G; pyrimidine, A/T) context, the overall mutational frequency of A or T bases was not affected. Moreover, the overall mutational frequency of the sequences examined was not decreased. There was an apparent increase in the number of insertions and deletions. The results are consistent with the conclusion that pol eta specifically targets WA motifs. However, its overall contribution to the somatic hypermutational process does not appear to be indispensable and in its absence other mechanisms maintain mutational activity.

FlashPlate scintillation proximity assays for characterization and screening of DNA polymerase, primase, and helicase activities.

DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [(3)H]-dNTPs onto the growing primer 3' end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [(3)H]-rNTPs or by subsequent polymerase extension with [(3)H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [(33)P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.

An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases.

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions. In combination with the scintillation proximity assay (SPA[trade]), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides. A proof of the concept is obtained using pseudo-thymidine (psiT), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psiT arising from the carbon-carbon bond between the sugar and the base make it an interesting probe for the importance of conformational restraints in the active site of polymerases during primer elongation. From a pool of commercially available thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides containing psiT. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psiT are presented. This is the first time that PCR has been performed with a C-nucleoside.