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1
Panjkovic, Milana, und Tatjana Ivkovic-Kapicl. „Etiology and pathogenesis of precancerous lesions and invasive cervical carcinoma“. Medical review 61, Nr. 7-8 (2008): 364–68. http://dx.doi.org/10.2298/mpns0808364p.
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Cervical cancer is the second most common gynecological malignancy in the world. Human papilloma virus (HPV) infection is the leading ethiologic agent in the development of premalignant and malignant cervical diseases. HPV is a member of the Papovaviridae family and until now over 100 types have been recognized. There are two types of viral infection: latent and productive. Virus induced oncogenesis is the result of interaction between virus oncoproteins E6 and E7 and tumor supresor host genes p53 and Rb. Many cofactors such as immunosuppression, early sexual relationship, multiple sexual partners, other sexualy transsmited infections and smooking are contributing factors of the precancerous and invasive cervical lesions. According to the oncogenic potential HPV are divided into three groups: low, intermediate and high oncogenic risk viruses. Molecular technics which are used for the virus detection are: In situ hibridisation,, Hybrid capture test and polymerasa chain reaction. Human papilloma virus testing has an important role in the follow up and treatment of women with 'atypical squamous cells of unknown significant' changes in cervical smears and low-grade squamous intraepithelial lesions, changes in punch biopsy.
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Vidić, Branka, Stanko Boboš, Sara Savić und Živoslav Grgić. „MYCOPLASMA AS THE CAUSE OF INFECTIONS IN CATTLE“. Archives of Veterinary Medicine 5, Nr. 2 (26.12.2012): 11–18. http://dx.doi.org/10.46784/e-avm.v5i2.166.
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During the last few years more than 20 different types of Mycoplasma, Ureaplasma and Acholeplasma microorganisms have been isolated from cattle with different clinical signs. Most of Mycoplasma microorganisms have a secondary role in the appearance of infection in cattle. Differently, Mycoplasma bovis (M. bovis) has a primary role in the infection of cattle. It has been proved that M. bovis frequently causes pneumonia, mastitis and arthritis in cows. Besides, M. bovis is identified as a causative agent in meningitis, middle ear infection, keratoconjunctivitis, decubitus abscesses, vaginitis and miscarriages in cows. Diagnostic was done based on the clinical signs and detection of the causative agent, in individual animals and in the herds as whole. The methods used for diagnostic can be cultivation of the causative agent or fluorescence test for antigen detection in pathological material. Also, the detection of specific antibodies can be done, applying different serological methods: fast agglutination on a plate, indirect hemiinhibition, agar gel immunodifussion, CF, ELISA, etc. Polymerasa chain reaction (PCR) is a sensitive method which is mostly used for confirmatory etiological diagnostic. The treatment of Mycoplasma infection is very demanding due to the resistance to most frequently used antibiotics and therefore very different from treating any other bacterial infection. M. bovis is one of the most complicated agents for control as its response to the treatment is weak. A good program for eradication of mycoplasmosis is based on early carrier detection and they are excluded the herds.
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Hili, Ryan, Chun Guo, Dehui Kong und Yi Lei. „Expanding the Chemical Diversity of DNA“. Synlett 29, Nr. 11 (20.03.2018): 1405–14. http://dx.doi.org/10.1055/s-0036-1591959.
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Nucleic acid polymers can be evolved to exhibit desired properties, including molecular recognition of a molecular target and catalysis of a specific reaction. These properties can be readily evolved despite the dearth of chemical diversity available to nucleic acid polymers, especially when compared to the rich chemical complexity of proteins. Expansion of nucleic acid chemical diversity has therefore been an important thrust for improving their properties for analytical and biomedical applications. Herein, we briefly describe the current state-of-the-art for the sequence-defined incorporation of modifications throughout an evolvable nucleic acid polymer. This includes contributions from our own lab, which have expanded the chemical diversity of nucleic acid polymers closer to the level observed in proteinogenic polymers.1 Introduction2 Polymerase-Catalyzed Synthesis of Modified Nucleic Acid Polymers3 Ligase-Catalyzed Oligonucleotide Polymerization (LOOPER)4 LOOPER with Small Modifications5 LOOPER with Large Modifications6 Evolution of Aptamers Derived from LOOPER Libraries7 Outlook
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Gibbs, J. S., K. Weisshart, P. Digard, A. deBruynKops, D. M. Knipe und D. M. Coen. „Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.“ Molecular and Cellular Biology 11, Nr. 9 (September 1991): 4786–95. http://dx.doi.org/10.1128/mcb.11.9.4786.
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Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.
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Gibbs, J. S., K. Weisshart, P. Digard, A. deBruynKops, D. M. Knipe und D. M. Coen. „Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site“. Molecular and Cellular Biology 11, Nr. 9 (September 1991): 4786–95. http://dx.doi.org/10.1128/mcb.11.9.4786-4795.1991.
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Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.
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Jadranin, Zeljko, Vesna Suljagic, Veljko Todorovic, Miroljub Trkuljic und Dusan Vucetic. „HIV/AIDS and other sexually transmitted infections among military members of the armed forces of Serbia“. Vojnosanitetski pregled 69, Nr. 1 (2012): 43–48. http://dx.doi.org/10.2298/vsp1201043j.
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Background/Aim. Military personnel is a population group at special risk of exposure to sexually transmitted diseases (STD). In peacetime, STD infection rates among service members are generally 2 to 5 times higher than among civilian population. In time of conflict, the differences can be 50 or more times greater. This study describes sexual behavior as a risk factor for STD in the Armed Forces of Serbia. Methods. The sample of 5 617 voluntary blood donors from the Armed Forces of Serbia gave blood and filled World Health Organization Questionnaire about sexual behavior within January 2007 - December 2008 period. The mandatory testing of voluntary blood donors was performed in the Institute of Transfusiology Military Medical Academy in Belgrade, by the specific immunoenzyme tests and polymerasa chain reaction tests for HIV, hepatitis B, C and syphilis. Statistical analysis of data was done using State for Windows 93, USA, 1996. Results. We identified 36 soldiers with some form of STDs. This study showed that 1 668 (29.7%) tested soldiers reported always using condoms, 1 725 (30.72%) almost always, 1 238 (20.04%) sometimes, 495 (8.81%) almost never and 490 (8.73%) never. Among the sample, 449 (7.99%) soldiers reported sexual contacts with partners with high risk of sexual behavior, whilst 22 (0.37%) of them reported homosexual and bisexual contacts. Conclusion. This study reported STDs found in voluntary blood donors among the service members of the Armed Forces of Serbia, but none of them was identified to be HIV positive. Soldiers with the most frequent risk behavior were reported to be those with inconsistent condom use. In the future, the STD Control and Prevention Program should be more intensively conducted among the members of the Armed Forces of Serbia.
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Raia, Pierre, Marc Delarue und Ludovic Sauguet. „An updated structural classification of replicative DNA polymerases“. Biochemical Society Transactions 47, Nr. 1 (15.01.2019): 239–49. http://dx.doi.org/10.1042/bst20180579.
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AbstractReplicative DNA polymerases are nano-machines essential to life, which have evolved the ability to copy the genome with high fidelity and high processivity. In contrast with cellular transcriptases and ribosome machines, which evolved by accretion of complexity from a conserved catalytic core, no replicative DNA polymerase is universally conserved. Strikingly, four different families of DNA polymerases have evolved to perform DNA replication in the three domains of life. In Bacteria, the genome is replicated by DNA polymerases belonging to the A- and C-families. In Eukarya, genomic DNA is copied mainly by three distinct replicative DNA polymerases, Polα, Polδ, and Polε, which all belong to the B-family. Matters are more complicated in Archaea, which contain an unusual D-family DNA polymerase (PolD) in addition to PolB, a B-family replicative DNA polymerase that is homologous to the eukaryotic ones. PolD is a heterodimeric DNA polymerase present in all Archaea discovered so far, except Crenarchaea. While PolD is an essential replicative DNA polymerase, it is often underrepresented in the literature when the diversity of DNA polymerases is discussed. Recent structural studies have shown that the structures of both polymerase and proofreading active sites of PolD differ from other structurally characterized DNA polymerases, thereby extending the repertoire of folds known to perform DNA replication. This review aims to provide an updated structural classification of all replicative DNAPs and discuss their evolutionary relationships, both regarding the DNA polymerase and proofreading active sites.
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Jung, G. H., M. C. Leavitt, J. C. Hsieh und J. Ito. „Bacteriophage PRD1 DNA polymerase: evolution of DNA polymerases.“ Proceedings of the National Academy of Sciences 84, Nr. 23 (01.12.1987): 8287–91. http://dx.doi.org/10.1073/pnas.84.23.8287.
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Walsh, Jason M., und Penny J. Beuning. „Synthetic Nucleotides as Probes of DNA Polymerase Specificity“. Journal of Nucleic Acids 2012 (2012): 1–17. http://dx.doi.org/10.1155/2012/530963.
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The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example,Escherichia coliDNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides.
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Miura, Masashi, Chihiro Tanigawa, Yoshito Fujii und Satoshi Kaneko. „COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR“. Revista do Instituto de Medicina Tropical de São Paulo 55, Nr. 6 (Dezember 2013): 401–6. http://dx.doi.org/10.1590/s0036-46652013000600005.
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SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.
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Choi, Jeong Jin, Jae-Geun Song, Ki Hoon Nam, Jong Il Lee, Heejin Bae, Gun A. Kim, Younguk Sun und Suk-Tae Kwon. „Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase“. Applied and Environmental Microbiology 74, Nr. 21 (12.09.2008): 6563–69. http://dx.doi.org/10.1128/aem.00624-08.
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ABSTRACT The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 � 10−6) than Taq DNA polymerase (11.98 � 10−6). Uniquely, Neq DNA polymerase also amplified λ DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.
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Davalieva, Katarina, und Georgi D. Efremov. „Influence of salts and pcr inhibitors on the amplification capacity of three thermostable DNA polymerases“. Macedonian Journal of Chemistry and Chemical Engineering 29, Nr. 1 (15.06.2010): 57. http://dx.doi.org/10.20450/mjcce.2010.173.
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The full potential of PCR as a rapid DNA detection method, is limited by inhibition of thermostable DNA polymerases by components in biological samples and substances used for purification of template DNA. We compared the inhibition effect of blood, phenol and various ions on Taq, Tth and Tne thermostable DNA polymerases prepared by us. The amplification capacity of DNA polymerases was tested on the amplification of a 631 bp fragment of a â- globin gene from a 100 ng DNA template under the optimal PCR buffer. Blood above 1 % (v/v) and phenol above 0.1 % (v/v) inhibited Taq, while Tne and Tth tolerated 10 to 30 times more. The inhibitory effect of ions is lowest for the Tth DNA polymerase, followed by Taq and Tne DNA polymerase. We conclude that the PCR inhibiting effect of some substances to Taq DNA polymerase can be eliminated by the use of a more resistant thermostable DNA polymerase, such as Tth or Tne DNA polymerases.
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Gening, L. V., S. A. Klincheva, A. Reshetnjak, A. P. Grollman und H. Miller. „RNA aptamers selected against DNA polymerase inhibit the polymerase activities of DNA polymerases and“. Nucleic Acids Research 34, Nr. 9 (17.05.2006): 2579–86. http://dx.doi.org/10.1093/nar/gkl326.
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Hałas, A., A. Ciesielski und J. Zuk. „Involvement of the essential yeast DNA polymerases in induced gene conversion.“ Acta Biochimica Polonica 46, Nr. 4 (31.12.1999): 862–72. http://dx.doi.org/10.18388/abp.1999_4107.
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In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.
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Gaillard, Richard K., Jennifer Barnard, Vincent Lopez, Paula Hodges, Eric Bourne, Lance Johnson, Marchelle I. Allen, Patrick Condreay, Wayne H. Miller und Lynn D. Condreay. „Kinetic Analysis of Wild-Type and YMDD Mutant Hepatitis B Virus Polymerases and Effects of Deoxyribonucleotide Concentrations on Polymerase Activity“. Antimicrobial Agents and Chemotherapy 46, Nr. 4 (April 2002): 1005–13. http://dx.doi.org/10.1128/aac.46.4.1005-1013.2002.
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ABSTRACT Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent Km values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased Ki values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent Km values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 μM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased Km values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.
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Budd, M. E., und J. L. Campbell. „DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae.“ Molecular and Cellular Biology 15, Nr. 4 (April 1995): 2173–79. http://dx.doi.org/10.1128/mcb.15.4.2173.
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The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled.
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LEHMANN, A., A. NIIMI, T. OGI, S. BROWN, S. SABBIONEDA, J. WING, P. KANNOUCHE und C. GREEN. „Translesion synthesis: Y-family polymerases and the polymerase switch“. DNA Repair 6, Nr. 7 (01.07.2007): 891–99. http://dx.doi.org/10.1016/j.dnarep.2007.02.003.
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McIntyre, Justyna. „Polymerase iota - an odd sibling among Y family polymerases“. DNA Repair 86 (Februar 2020): 102753. http://dx.doi.org/10.1016/j.dnarep.2019.102753.
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Cohen, Susan E., Veronica G. Godoy und Graham C. Walker. „Transcriptional Modulator NusA Interacts with Translesion DNA Polymerases in Escherichia coli“. Journal of Bacteriology 191, Nr. 2 (07.11.2008): 665–72. http://dx.doi.org/10.1128/jb.00941-08.
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ABSTRACT NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.
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Harada, Ryo, Yoshihisa Hirakawa, Akinori Yabuki, Yuichiro Kashiyama, Moe Maruyama, Ryo Onuma, Petr Soukal et al. „Inventory and Evolution of Mitochondrion-localized Family A DNA Polymerases in Euglenozoa“. Pathogens 9, Nr. 4 (01.04.2020): 257. http://dx.doi.org/10.3390/pathogens9040257.
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The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, “plant and protist organellar DNA polymerase (POP)” in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa—Kinetoplastea, Diplonemea, and Euglenida—to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida.
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Coello-Coutiño, P., E. García-Ramírez und J. M. Vázquez-Ramos. „Preparation of an antibody against a maize DNA polymerase holoenzyme: identification of the polymerase catalytic subunit“. Canadian Journal of Botany 72, Nr. 6 (01.06.1994): 818–22. http://dx.doi.org/10.1139/b94-104.
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Three different DNA polymerase activities can be separated from germinating maize axes through DEAE – cellulose chromatography. Of these, DNA polymerase 2 appears to be a replicative-type enzyme composed of several subunits. An antibody has been developed against the DNA polymerase 2 multisubunit complex, which mainly recognizes a polypeptide of molecular weight around 90 kDa. Polypeptides of molecular mass of 83, 70, 60, 55, 45, and 24 kDa are also recognized. Activity gels showed that the 90-kDa polypeptide possesses catalytic activity. DNA polymerases 1 and 3 are not recognized by the antibody and their activities are not reduced. However, DNA polymerase 2 activity is reduced by 70%. The nature of the different DNA polymerase accompanying subunits is discussed. Key words: DNA polymerases, maize embryo axes.
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Thomsen, Darrell R., Nancee L. Oien, Todd A. Hopkins, Mary L. Knechtel, Roger J. Brideau, Michael W. Wathen und Fred L. Homa. „Amino Acid Changes within Conserved Region III of the Herpes Simplex Virus and Human Cytomegalovirus DNA Polymerases Confer Resistance to 4-Oxo-Dihydroquinolines, a Novel Class of Herpesvirus Antiviral Agents“. Journal of Virology 77, Nr. 3 (01.02.2003): 1868–76. http://dx.doi.org/10.1128/jvi.77.3.1868-1876.2003.
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ABSTRACT The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.
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Bettiol, Michael F., Randall T. Irvin und Paul A. Horgen. „Immunological analyses of selected eukaryotic RNA polymerases II“. Canadian Journal of Biochemistry and Cell Biology 63, Nr. 12 (01.12.1985): 1217–30. http://dx.doi.org/10.1139/o85-153.
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Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.
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Maul, Robert W., und Mark D. Sutton. „Roles of the Escherichia coli RecA Protein and the Global SOS Response in Effecting DNA Polymerase Selection In Vivo“. Journal of Bacteriology 187, Nr. 22 (15.11.2005): 7607–18. http://dx.doi.org/10.1128/jb.187.22.7607-7618.2005.
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ABSTRACT The Escherichia coli β sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the dnaN159 allele, which encodes a mutant form of the β clamp (β159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in E. coli, we have further characterized effects of the dnaN159 allele on polymerase usage. Three of the five E.coli DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the dinB-encoded polymerase IV is sufficient to result in conditional lethality of the dnaN159 strain. In contrast, chronically activated RecA protein, expressed from the recA730 allele, is lethal to the dnaN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., ΔrecR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., ΔpolB or ΔumuDC). Thus, we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the dnaN159 strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA polymerases are managed in vivo.
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Jensen, Grant J., Gavin Meredith, David A. Bushnell und Roger D. Kornberg. „Structure of Wild Type Yeast RNA Polymerase II and Location of RPB4 and RPB7“. Microscopy and Microanalysis 4, S2 (Juli 1998): 972–73. http://dx.doi.org/10.1017/s1431927600024983.
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Nucleic acid polymerase structure has been studied by both X-ray and electron crystallography. To date, only the smaller, single subunit polymerases have been subjected to X-ray analysis, including the bacteriophage T7 RNA polymerase, which is the only RNA polymerase whose structure is known to atomic resolution. Lower resolution structures of several multisubunit polymerases have been determined by electron crystallography, including a mutant form of yeast RNA polymerase II which lacks subunits Rpb4 and Rpb7 (denoted A4/7 polymerase). All polymerase structures obtained by both X-ray and electron crystallography show a large cleft appropriate in size for binding duplex DNA, and further appear to contain a mobile arm allowing open and closed conformations of the cleft, presumably permitting entry and retention of DNA. Subunits Rpb4 and Rpb7 of RNA polymerase II form a dissociable subcomplex that has been implicated in the stress response and in the initiation of transcription. Human homologs of Rpb4 and Rpb7 have been identified.
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Crotty, Shane, David Gohara, Devin K. Gilligan, Sveta Karelsky, Craig E. Cameron und Raul Andino. „Manganese-Dependent Polioviruses Caused by Mutations within the Viral Polymerase“. Journal of Virology 77, Nr. 9 (01.05.2003): 5378–88. http://dx.doi.org/10.1128/jvi.77.9.5378-5388.2003.
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ABSTRACT Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3Dpol, we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn2+-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.
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Murphy, Kelly, Hariyanto Darmawan, Amy Schultz, Elizabeth Fidalgo da Silva und Linda J. Reha-Krantz. „A method to select for mutator DNA polymerase δs in Saccharomyces cerevisiae“. Genome 49, Nr. 4 (01.04.2006): 403–10. http://dx.doi.org/10.1139/g05-106.
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Proofreading DNA polymerases share common short peptide motifs that bind Mg2+ in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase δ mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase δ mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.Key words: DNA polymerase proofreading, genetic selection for mutator mutants, fidelity of DNA replication, yeast.
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Bridges, B. A., Helen Bates und Firdaus Sharif. „Polymerases and UV mutagenesis in Escherichia coli“. Genome 31, Nr. 2 (15.01.1989): 572–77. http://dx.doi.org/10.1139/g89-106.
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Evidence for and against the involvement of the known nucleic acid polymerases in UV mutagenesis in Escherichia coli is reviewed. There is no evidence that rules out the participation of any of them when they are present but only one, the α subunit of DNA polymerase III holoenzyme (polC gene product) has been shown to be essential. It is argued that the PolC protein that functions in UV mutagenesis may not be immediately recognizable as one of the normal cellular polymerases or polymerase complexes.Key words: polymerases, ultraviolet light, mutagenesis, DNA repair, misincorporation.
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McInerney, Peter, Paul Adams und Masood Z. Hadi. „Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase“. Molecular Biology International 2014 (17.08.2014): 1–8. http://dx.doi.org/10.1155/2014/287430.
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As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.
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Egorova, Tatiana, Ekaterina Shuvalova, Sabina Mukba, Alexey Shuvalov, Peter Kolosov und Elena Alkalaeva. „Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides“. International Journal of Molecular Sciences 22, Nr. 10 (14.05.2021): 5186. http://dx.doi.org/10.3390/ijms22105186.
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Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.
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Pavlov, Youri I., Anna S. Zhuk und Elena I. Stepchenkova. „DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer“. Cancers 12, Nr. 12 (24.11.2020): 3489. http://dx.doi.org/10.3390/cancers12123489.
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Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. The principal idea of participation of different polymerases in specific transactions at the fork proposed by Morrison and coauthors 30 years ago and later named “division of labor,” remains standing, with an amendment of the broader role of polymerase δ in the replication of both the lagging and leading DNA strands. However, cancer-associated mutations predominantly affect the catalytic subunit of polymerase ε that participates in leading strand DNA synthesis. We analyze how new findings in the DNA replication field help elucidate the polymerase variants’ effects on cancer.
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Core, Leighton J., Joshua J. Waterfall und John T. Lis. „Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters“. Science 322, Nr. 5909 (04.12.2008): 1845–48. http://dx.doi.org/10.1126/science.1162228.
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RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on ∼30% of human genes, transcription extends beyond pre-messenger RNA 3′ cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
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Ji, Junwei, und Anil Day. „Construction of a highly error-prone DNA polymerase for developing organelle mutation systems“. Nucleic Acids Research 48, Nr. 21 (02.11.2020): 11868–79. http://dx.doi.org/10.1093/nar/gkaa929.
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Abstract A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.
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McDonald, John P., Agnès Tissier, Ekaterina G. Frank, Shigenori Iwai, Fumio Hanaoka und Roger Woodgate. „DNA polymerase iota and related Rad30–like enzymes“. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 356, Nr. 1405 (29.01.2001): 53–60. http://dx.doi.org/10.1098/rstb.2000.0748.
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Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so–called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase η and defects in the protein lead to the xeroderma pigmentosum variant (XP–V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol ι. Based upon in vitro studies, it appears that Pol ι has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error–prone DNA polymerase.
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Pan, Junhua, Vikram N. Vakharia und Yizhi Jane Tao. „The structure of a birnavirus polymerase reveals a distinct active site topology“. Proceedings of the National Academy of Sciences 104, Nr. 18 (24.04.2007): 7385–90. http://dx.doi.org/10.1073/pnas.0611599104.
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Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.
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Pospiech, Helmut, und Juhani E. Syväoja. „DNA Polymerase e - More Than a Polymerase“. Scientific World JOURNAL 3 (2003): 87–104. http://dx.doi.org/10.1100/tsw.2003.08.
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This paper presents a comprehensive review of the structure and function of DNA polymerase e. Together with DNA polymerases a and d, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase a lays down RNA-DNA primers that are utilized by DNA polymerases d and e for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase e does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase e fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell.
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Budd, M. E., und J. L. Campbell. „DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.“ Molecular and Cellular Biology 13, Nr. 1 (Januar 1993): 496–505. http://dx.doi.org/10.1128/mcb.13.1.496.
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Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.
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Budd, M. E., und J. L. Campbell. „DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae“. Molecular and Cellular Biology 13, Nr. 1 (Januar 1993): 496–505. http://dx.doi.org/10.1128/mcb.13.1.496-505.1993.
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Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.
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Niimi, Atsuko, Siripan Limsirichaikul, Shonen Yoshida, Shigenori Iwai, Chikahide Masutani, Fumio Hanaoka, Eric T. Kool, Yukihiro Nishiyama und Motoshi Suzuki. „Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity“. Molecular and Cellular Biology 24, Nr. 7 (01.04.2004): 2734–46. http://dx.doi.org/10.1128/mcb.24.7.2734-2746.2004.
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ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.
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Villarreal, Luis P., und Victor R. DeFilippis. „A Hypothesis for DNA Viruses as the Origin of Eukaryotic Replication Proteins“. Journal of Virology 74, Nr. 15 (01.08.2000): 7079–84. http://dx.doi.org/10.1128/jvi.74.15.7079-7084.2000.
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ABSTRACT The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.
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Steitz, T., S. Smerdon, J. Jager und C. Joyce. „A unified polymerase mechanism for nonhomologous DNA and RNA polymerases“. Science 266, Nr. 5193 (23.12.1994): 2022–25. http://dx.doi.org/10.1126/science.7528445.
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42
Leavitt, M. C., und J. Ito. „T5 DNA polymerase: structural--functional relationships to other DNA polymerases.“ Proceedings of the National Academy of Sciences 86, Nr. 12 (01.06.1989): 4465–69. http://dx.doi.org/10.1073/pnas.86.12.4465.
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Cann, Isaac K. O., Sonoko Ishino, Norimichi Nomura, Yoshihiko Sako und Yoshizumi Ishino. „Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote“. Journal of Bacteriology 181, Nr. 19 (01.10.1999): 5984–92. http://dx.doi.org/10.1128/jb.181.19.5984-5992.1999.
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ABSTRACT DNA polymerase activities in fractionated cell extract ofAeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3′→5′ exonuclease and polymerase activities. The product ofpolA gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II ofPyrodictium occultum. The deduced amino acid sequence ofA. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomainCrenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.
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Kawaguchi, Atsushi, Tadasuke Naito und Kyosuke Nagata. „Involvement of Influenza Virus PA Subunit in Assembly of Functional RNA Polymerase Complexes“. Journal of Virology 79, Nr. 2 (15.01.2005): 732–44. http://dx.doi.org/10.1128/jvi.79.2.732-744.2005.
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ABSTRACT The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear. ts53 virus is a temperature-sensitive (ts) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology 65:363-373, 1975). We confirmed that the mRNA synthesis level of ts53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding ts53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In ts53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.
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Steitz, Thomas A., und Y. Whitney Yin. „Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases“. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, Nr. 1441 (29.01.2004): 17–23. http://dx.doi.org/10.1098/rstb.2003.1374.
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The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active–site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain α–helices by 60° upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40° and the thumb domain re–orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non–template strand from the template strand.
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Hockman, D. J., und M. C. Schultz. „Casein kinase II is required for efficient transcription by RNA polymerase III.“ Molecular and Cellular Biology 16, Nr. 3 (März 1996): 892–98. http://dx.doi.org/10.1128/mcb.16.3.892.
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Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature-sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III.
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French, Sarah L., Yvonne N. Osheim, David A. Schneider, Martha L. Sikes, Cesar F. Fernandez, Laura A. Copela, Vikram A. Misra, Masayasu Nomura, Sandra L. Wolin und Ann L. Beyer. „Visual Analysis of the Yeast 5S rRNA Gene Transcriptome: Regulation and Role of La Protein“. Molecular and Cellular Biology 28, Nr. 14 (12.05.2008): 4576–87. http://dx.doi.org/10.1128/mcb.00127-08.
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ABSTRACT 5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, ∼132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of ∼1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.
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Zhang, Jiayou. „Host RNA polymerase II makes minimal contributions to retroviral frame-shift mutations“. Journal of General Virology 85, Nr. 8 (01.08.2004): 2389–95. http://dx.doi.org/10.1099/vir.0.80081-0.
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The rate of mutation during retrovirus replication is high. Mutations can occur during transcription of the viral genomic RNA from the integrated provirus or during reverse transcription from viral RNA to form viral DNA or during replication of the proviral DNA as the host cell is dividing. Therefore, three polymerases may all contribute to retroviral evolution: host RNA polymerase II, viral reverse transcriptases and host DNA polymerases, respectively. Since the rate of mutation for host DNA polymerase is very low, mutations are more likely to be caused by the host RNA polymerase II and/or the viral reverse transcriptase. A system was established to detect the frequency of frame-shift mutations caused by cellular RNA polymerase II, as well as the rate of retroviral mutation during a single cycle of replication in vivo. In this study, it was determined that RNA polymerase II contributes less than 3 % to frame-shift mutations that occur during retrovirus replication. Therefore, the majority of frame-shift mutations detected within the viral genome are the result of errors during reverse transcription.
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Campagnola, Grace, Mark Weygandt, Kirsten Scoggin und Olve Peersen. „Crystal Structure of Coxsackievirus B3 3Dpol Highlights the Functional Importance of Residue 5 in Picornavirus Polymerases“. Journal of Virology 82, Nr. 19 (16.07.2008): 9458–64. http://dx.doi.org/10.1128/jvi.00647-08.
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ABSTRACT The crystal structure of the coxsackievirus B3 polymerase has been solved at 2.25-Å resolution and is shown to be highly homologous to polymerases from poliovirus, rhinovirus, and foot-and-mouth disease viruses. Together, these structures highlight several conserved structural elements in picornaviral polymerases, including a proteolytic activation-dependent N-terminal structure that is essential for full activity. Interestingly, a comparison of all of the picornaviral polymerase structures shows an unusual conformation for residue 5, which is always located at a distortion in the β-strand composed of residues 1 to 8. In our earlier structure of the poliovirus polymerase, we attributed this conformation to a crystal packing artifact, but the observation that this conformation is conserved among picornaviruses led us to examine the role of this residue in further detail. Here we use coxsackievirus polymerase to show that elongation activity correlates with the hydrophobicity of residue 5 and, surprisingly, more hydrophobic residues result in higher activity. Based on structural analysis, we propose that this residue becomes buried during the nucleotide repositioning step that occurs prior to phosphoryl transfer. We present a model in which the buried N terminus observed in all picornaviral polymerases is essential for stabilizing the structure during this conformational change.
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Beckman, Robert A., und Lawrence A. Loeb. „Multi-stage proofreading in DNA replication“. Quarterly Reviews of Biophysics 26, Nr. 3 (August 1993): 225–331. http://dx.doi.org/10.1017/s0033583500002869.
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The mechanisms by which DNA polymerases achieve their remarkable fidelity, including base selection and proofreading, are briefly reviewed. Nine proofreading models from the current literature are evaluated in the light of steady-state and transient kinetic studies of E. coli DNA polymerase I, the beststudied DNA polymerase.