In Vitro Identification of Phosphorylation Sites on TcPolß by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolß in Trypanosoma cruzi Epimastigotes.

Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolß) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolß by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolß. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolß. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolß phosphorylation and enzymatic activity in T. cruzi epimastigotes.

Quantitative profiling of Marek's Disease Virus in vaccinated layer chicken.

The present study was undertaken to quantify the Marek's Disease Virus (MDV) serotypes in vaccinated commercial layer flocks at 7, 14, 21, 28, 35 and 60-90 days post vaccination (dpv) and to correlate the pathogenic Gallid herpesvirus 2 (GaHV-2, MDV1) load with vaccine viral load of Gallid herpesvirus 3 (GaHV-3, MDV2) and Meleagridis herpesvirus 1 (MeHV-1, MDV3). A total of 25 commercial layer flocks were selected in and around Namakkal district of Tamil nadu, India and the feather pulp (FP) and blood samples were collected. Out of 25 flocks, 14 were revaccinated with bivalent vaccine, six were revaccinated with monovalent vaccine apart from the initial bivalent vaccination done at hatchery and five flocks were not revaccinated. SYBR green based real time PCR was used for absolute quantification of MDV serotypes. The pathogenic MDV1 load had shown an increasing trend until 21 dpv followed by a dip and again had shown a constant uptick between 60 and 90 dpv in the flocks that went on to develop MD outbreak. The flocks which had not encountered any Marek's Disease outbreak had shown increasing trend of MDV2 and 3 load until 21 dpv followed by a slight decrease but maintained a higher load when compared to MDV 1 which had marked a sharp decline between 60 and 90 dpv. Outbreak of MD was observed in seven (28%) out of 25 flocks between 18 and 27 weeks of age. It includes, two out of fourteen farms (14%) revaccinated with bivalent vaccine, two out of six farms (33%) revaccinated with MDV3 vaccine and three out of five farms (60%) without revaccination. The overall mean of vaccine viral load at various stages of dpv was constantly low where as pathogenic MDV 1 load was constantly high between 60 and 90 dpv in the flocks that went on to develop Marek's Disease during later part of life.

Underappreciated Roles of DNA Polymerase δ in Replication Stress Survival.

Recent structural analysis of Fe-S centers in replication proteins and insights into the structure and function of DNA polymerase δ (DNA Pol δ) subunits have shed light on the key role played by this polymerase at replication forks under stress. The sequencing of cancer genomes reveals multiple point mutations that compromise the activity of POLD1, the DNA Pol δ catalytic subunit, whereas the loci encoding the accessory subunits POLD2 and POLD3 are amplified in a very high proportion of human tumors. Consistently, DNA Pol δ is key for the survival of replication stress and is involved in multiple long-patch repair pathways. Synthetic lethality arises from compromising the function and availability of the noncatalytic subunits of DNA Pol δ under conditions of replication stress, opening the door to novel therapies.

The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation.

The tripartite motif (TRIM) protein family is an E3 ubiquitin ligase family. Recent reports have indicated that some TRIM proteins have antiviral functions, especially against retroviruses. However, most studies mainly focus on the relationship between TRIM21 and interferon or other antiviral effectors. The effect of TRIM21 on virus-encoded proteins remains unclear. In this study, we screened candidate interacting proteins of HBV DNA polymerase (Pol) by FLAG affinity purification and mass spectrometry assay and identified TRIM21 as its regulator. We used a coimmunoprecipitation (co-IP) assay to demonstrate that TRIM21 interacted with the TP domain of HBV DNA Pol. In addition, TRIM21 promoted the ubiquitination and degradation of HBV DNA Pol using its RING domain, which has E3 ubiquitin ligase activity. Lys260 and Lys283 of HBV DNA Pol were identified as targets for ubiquitination mediated by TRIM21. Finally, we uncovered that TRIM21 degrades HBV DNA Pol to restrict HBV DNA replication, and its SPRY domain is critical for this activity. Taken together, our results indicate that TRIM21 suppresses HBV DNA replication mainly by promoting the ubiquitination of HBV DNA Pol, which may provide a new potential target for the treatment of HBV.

Replication fidelity in E. coli: Differential leading and lagging strand effects for dnaE antimutator alleles.

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.

Human DNA polymerase η has reverse transcriptase activity in cellular environments.

Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.

Subunits of the DNA polymerase alpha-primase complex promote Notch-mediated proliferation with discrete and shared functions in C. elegans germline.

Notch receptor signaling is a highly conserved cell communication system in most multicellular organisms and plays a critical role at several junctures in animal development. In Caenorhabditis elegans,GLP-1/Notch signaling is essential for both germline stem cell maintenance and germ cell proliferation during gonad development. Here, we show that subunits (POLA-1, DIV-1, PRI-1, and PRI-2) of the DNA polymerase alpha-primase complex are required for germ cell proliferation in response to GLP-1/Notch signaling in different tissues at different developmental stages. Specifically, genetic and functional analyses demonstrated that (a) maternally contributed DIV-1 (regulatory subunit) is indispensable non-cell autonomously for GLP-1/Notch-mediated germ cell proliferation during early larval development, whereas POLA-1 (catalytic subunit) and two primase subunits, PRI-1 and PRI-2, do not appear to be essential; (b) germline POLA-1, PRI-1, and PRI-2 play a crucial role in GLP-1/Notch-mediated maintenance of proliferative cell fate during adulthood, while DIV-1 is dispensable; and (c) germline POLA-1, DIV-1, PRI-1, and PRI-2 function in tandem with PUF (Pumilio/FBF) RNA-binding proteins to maintain germline stem cells in the adult gonad. These findings suggest that the subunits of the DNA polymerase alpha-primase complex exhibit both discrete and shared functions in GLP-1/Notch or PUF-mediated germ cell dynamics in C. elegans. These findings link the biological functions of DNA replication machineries to signals that maintain a stem cell population, and may have further implications for Notch-dependent tumors.

Targeted mutagenesis: A sniper-like diversity generator in microbial engineering.

Mutations, serving as the raw materials of evolution, have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions. Global and targeted mutagenesis are two main methods of obtaining various mutations, distinguished by the range of action they can cover. While the former one stresses the mining of novel genetic loci within the whole genomic background, targeted mutagenesis performs in a more straightforward manner, bringing evolutionary escape and error catastrophe under control. In this review, we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo, and briefly introduce the mechanisms and applications of them separately. The inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.

Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein-protein interactions.

Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD', form UmuD'2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine-thymine dimers, whereas DinB is specialized to copy N(2)-dG adducts, such as N(2)-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N(2)-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9V, H282P, and T412I, to bypass N(2)-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9V to render cells UV-mutable is dependent on the proper length of the arm of UmuD'. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD', which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.

Alternariol enhanced DNA polymerase ? expression in NIH3T3 cells / 基础医学与临床

Objective To study the effects of Alternariol(AOH) on DNA polymerase ?(DNA POL?)expression in NIH3T3 cells.Methods RT-PCR,Immunocytochemistry and Western blot were used to detected mRNA and the protein levels of DNA POL? in NIH3T3 cell line induced by AOH.Results The expression of DNA POL? in NIH3T3 cells contaminated by AOH was significantly higher than that in the control group(P

Establishment of human lung adenocarcinoma multidrug resistance cell lines induced by paclitaxel and the mRNA expressions of DNA pol?,mdr1,mrp1,GST-?,lrp,and topo Ⅱ genes / 西安交通大学学报(医学版)

Objective To establish human lung adenocarcinoma multidrug resistance cell lines in vitro,observe their biological characteristics,and investigate the mRNA expressions of DNA pol?,mdr 1,mrp1,GST-?,lrp and topo Ⅱ genes.Methods Paclitaxel-resistant cell lines(A549/TXL20) were established in vitro by exposure to stepwise increased concentrations of the drug in a cell culture medium.Biological morphology and cell cycles were analyzed by morphometry and flow cytometry.The chemoresistance indexes of cells were measured by methyl tetrazolium assay.Evaluation of growth and in vitro drug sensitivity were performed.RT-PCR was employed to analyze the mRNA expressions of the DNA pol?,mdr 1,mrp1,GST-?,lrp,and topo Ⅱ genes.Results ① Compared with parent cells,the resistant sublines had a lower confluent density.They were smaller and mixed with giant cells in different sizes and with different numbers of nucleoli,and the growth property of A549/TXL20 did not change significantly compared with A549 cell lines.② The resistant cells,A549/TXL20,were 19.3 times more resistant to paclitaxel and 67.4 times more resistant to cisplatin than the parent cells,and also demonstrated cross-resistance to mitomycin,vinblastine,and 5-fluouracil(5-FU). ③ Compared with the A549 celllines,an unreasonably higher level of drug resistance and lower drug concentration was detected in A549/TXL20 cells after exposure to the drug in the culture medium.④ The mRNA expression level of DNA pol?,mdr1,GST-?,mrp1 andlrp genes in A549/TXL20 cells was significantly higher than that in A549 cell lines(P