Journal articles: 'DNA-directed DNA polymerase'

1

Nick McElhinny, Stephanie A., and Dale A. Ramsden. "Polymerase Mu Is a DNA-Directed DNA/RNA Polymerase." Molecular and Cellular Biology 23, no. 7 (April 1, 2003): 2309–15. http://dx.doi.org/10.1128/mcb.23.7.2309-2315.2003.

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ABSTRACT DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol μ), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol μ has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol β, although pol μ's substrate specificity is similar to that of pol β in most other respects. Moreover, pol μ more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol μ frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.

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2

Gloeckner, Christian, Ramon Kranaster, and Andreas Marx. "Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries." Current Protocols in Chemical Biology 2, no. 2 (April 2010): 89–109. http://dx.doi.org/10.1002/9780470559277.ch090183.

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3

Wright, G. E. "Nucleotide probes of DNA polymerases." Acta Biochimica Polonica 43, no. 1 (March 31, 1996): 115–24. http://dx.doi.org/10.18388/abp.1996_4522.

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The modified nucleotides, N2-(p-n-butylphenyl)dGTP and 2-(p-n-butylanilino) dATP and related compounds have been developed as inhibitor-probes of B family DNA polymerases. Synthetic approaches to these compounds are summarized. The nucleotides are potent, non-substrate inhibitors of DNA polymerase a. In contrast, they inhibit other members of the family with less potency but act as substrates for these enzymes. Modelling of the inhibitor: enzyme binding mechanism has been done based on the known structure of E. coli DNA polymerase I, and site-directed mutagenesis experiments to evaluate this mechanism are proposed.

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Adereth, Yair, Kristen J. Champion, Tien Hsu, and Vincent Dammai. "Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase." BioTechniques 38, no. 6 (June 2005): 864–68. http://dx.doi.org/10.2144/05386bm03.

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5

Saiki, R., D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis, and H. Erlich. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science 239, no. 4839 (January 29, 1988): 487–91. http://dx.doi.org/10.1126/science.2448875.

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Saiki, RK, DH Gelfand, S. Stoffel, SJ Scharf, R. Higuchi, GT Horn, KB Mullis, and HA Erlich. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science 239, no. 4839 (January 29, 1988): 487–91. http://dx.doi.org/10.1126/science.239.4839.487.

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7

CHANG, GWONG-JEN J., BARBARA J. B. JOHNSON, and DENNIS W. TRENT. "Site-Specific Oligonucleotide-Directed Mutagenesis Using T4 DNA Polymerase." DNA 7, no. 3 (April 1988): 211–17. http://dx.doi.org/10.1089/dna.1988.7.211.

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8

Zwierzchowski, L., W. Niedbalski, and D. Kleczkowska. "Effect of prolactin, progesterone, pregnancy and lactation on DNA synthesis and DNA polymerase activities in rabbit mammary gland." Journal of Endocrinology 114, no. 1 (July 1987): 139–45. http://dx.doi.org/10.1677/joe.0.1140139.

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ABSTRACT DNA synthesis and DNA polymerase-α, -β and -γ activities in the rabbit mammary gland were studied during hormone-directed cellular growth. It was found that during pregnancy, early lactation and after injection of prolactin, changes in the activity of DNA polymerase-α paralleled the rate of mammary gland DNA synthesis. It was also found that the amount of polymerase-α activity bound to isolated chromatin depended on the physiological state of the animal. During pregnancy and early lactation changes in the activity of chromatin-bound enzyme correlated directly with the rate of DNA synthesis (r = 0·83). Moreover, in virgin rabbits treated with prolactin the activity of chromatin-bound DNA polymerase-α increased markedly at the same time as the DNA-synthetic rate increased. No correlation of the DNA-synthetic rate was found with the activity of soluble (cytosolic) DNA polymerase-α or with the activity of soluble or chromatin-bound DNA polymerases-β and -γ. On the basis of these results it is suggested that in the developing mammary gland both the activity and cellular distribution of DNA polymerase-α might be subject to hormonal regulation. J. Endocr. (1987) 114, 139–145

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9

Sinha, Surajit, Paul H. Kim, and Christopher Switzer. "2‘,5‘-Linked DNA Is a Template for Polymerase-Directed DNA Synthesis." Journal of the American Chemical Society 126, no. 1 (January 2004): 40–41. http://dx.doi.org/10.1021/ja034986z.

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10

Lelyveld, Victor S., Wen Zhang, and Jack W. Szostak. "Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase." Proceedings of the National Academy of Sciences 117, no. 13 (March 18, 2020): 7276–83. http://dx.doi.org/10.1073/pnas.1922400117.

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All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3′-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3′-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3′→P5′ phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3′-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

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11

LOYA, Shoshana, Amira RUDI, Yoel KASHMAN, and Amnon HIZI. "Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases." Biochemical Journal 362, no. 3 (March 8, 2002): 685–92. http://dx.doi.org/10.1042/bj3620685.

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Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases α and β and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (Ki = 0.51μM and Ki = 0.53μM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby Km remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

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12

Torgersen, Helge, Dieter Blaas, and Tim Skern. "Low cost apparatus for primer-directed DNA amplification using Thermus aquaticus-DNA polymerase." Analytical Biochemistry 176, no. 1 (January 1989): 33–35. http://dx.doi.org/10.1016/0003-2697(89)90268-6.

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13

Shirai, T., and M. Go. "RNase-like domain in DNA-directed RNA polymerase II." Proceedings of the National Academy of Sciences 88, no. 20 (October 15, 1991): 9056–60. http://dx.doi.org/10.1073/pnas.88.20.9056.

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14

LOYA, Shoshana, Amira RUDI, Yoel KASHMAN, and Amnon HIZI. "Polycitone A, a novel and potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases." Biochemical Journal 344, no. 1 (November 8, 1999): 85–92. http://dx.doi.org/10.1042/bj3440085.

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Polycitone A, an aromatic alkaloid isolated from the ascidian Polycitorsp. exhibits potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. The drug inhibits retroviral reverse transcriptase (RT) [i.e. of human immunodeficiency virus type 1 (HIV), murine leukaemia virus (MLV) and mouse mammary tumour virus (MMTV)] as efficiently as cellular DNA polymerases (i.e. of both DNA polymerases α and β and Escherichia coliDNA polymerase I). The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function, on the other hand, is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the substrate binding site, since this inhibitor behaves competitively with respect to dTTP with poly(rA)˙oligo(dT) as template primer. Chemical transformations of the five phenol groups of polycitone A by methoxy groups have a determinant effect on the inhibitory potency. Thus, the pentamethoxy derivative which is devoid of all hydroxy moieties, loses significantly, by 40-fold, the ability to inhibit the DNA polymerase function. Furthermore, this analogue lacks the ability to inhibit DNA primer extension as well as the formation of the RT-DNA complex. Indeed, inhibition of the first step in DNA polymerization, the formation of the RT-DNA complex, and hence, of the overall process, could serve as a model for a universal inhibitor of the superfamily of DNA polymerases.

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15

Carver,, Theodore E., and David P. Millar. "Recognition of Sequence-Directed DNA Structure by the Klenow Fragment of DNA Polymerase I†." Biochemistry 37, no. 7 (February 1998): 1898–904. http://dx.doi.org/10.1021/bi9720843.

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16

Samson, Camille, Pierre Legrand, Mustafa Tekpinar, Jef Rozenski, Mikhail Abramov, Philipp Holliger, Vitor B. Pinheiro, Piet Herdwijn, and Marc Delarue. "Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution." Biomolecules 10, no. 12 (December 8, 2020): 1647. http://dx.doi.org/10.3390/biom10121647.

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Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

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17

Boyd, Mark T., Brian Foley, and Isadore Brodsky. "Evidence for Copurification of HERV-K–Related Transcripts and a Reverse Transcriptase Activity in Human Platelets From Patients With Essential Thrombocythemia." Blood 90, no. 10 (November 15, 1997): 4022–30. http://dx.doi.org/10.1182/blood.v90.10.4022.

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Abstract We have previously reported that particles resembling retroviral particles and possessing an RNA-directed DNA polymerase activity can be prepared from platelets. Furthermore, we and others have shown that these particles are present at higher levels in patients with essential thrombocythemia and polycythemia vera. We show here that these particles package RNA molecules that encode HERV-K–related pol genes. A subset of the RNA molecules that are packaged are likely to encode the RNA directed DNA polymerase activity and, because these RNAs possess long/full-length open reading frames for the reverse transcriptase and RNaseH (also for part of the integrase domains in genomic clones) of HERV-K, we propose that these transcripts are indeed strong candidates for encoding the enzyme activity found in these particles. Moreover, by using a modification of the polymerase chain reaction-based reverse transcriptase assay in which activated DNA is added during cDNA synthesis to suppress DNA polymerase-mediated RNA-directed DNA synthesis, we have found that the particle-associated enzyme behaves like a retroviral reverse transcriptase, further supporting the conclusion that retrovirus-like, perhaps HERV-K sequences, encode this enzyme activity.

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18

Liu, Huanting, James H. Naismith, and Ronald T. Hay. "Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase." Journal of Virology 74, no. 24 (December 15, 2000): 11681–89. http://dx.doi.org/10.1128/jvi.74.24.11681-11689.2000.

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ABSTRACT Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase α family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3′-5′-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087–1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.

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19

Strerath, Michael, Christian Gloeckner, Dan Liu, Andreas Schnur, and Andreas Marx. "Directed DNA Polymerase Evolution: Effects of Mutations in Motif C on the Mismatch-Extension Selectivity ofThermus aquaticus DNA Polymerase." ChemBioChem 8, no. 4 (March 5, 2007): 395–401. http://dx.doi.org/10.1002/cbic.200600337.

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20

Kanno, Tatsuo, Bruno Huettel, M. Florian Mette, Werner Aufsatz, Estelle Jaligot, Lucia Daxinger, David P. Kreil, Marjori Matzke, and Antonius J. M. Matzke. "Atypical RNA polymerase subunits required for RNA-directed DNA methylation." Nature Genetics 37, no. 7 (May 29, 2005): 761–65. http://dx.doi.org/10.1038/ng1580.

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21

Tsurusluta, Naoya, Hisaji Maki, and Laurence Jay Korn. "Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme." Gene 62, no. 1 (February 1988): 135–39. http://dx.doi.org/10.1016/0378-1119(88)90587-2.

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22

Nair, Deepak T., Robert E. Johnson, Louise Prakash, Satya Prakash, and Aneel K. Aggarwal. "Protein-Template-Directed Synthesis across an Acrolein-Derived DNA Adduct by Yeast Rev1 DNA Polymerase." Structure 16, no. 2 (February 2008): 239–45. http://dx.doi.org/10.1016/j.str.2007.12.009.

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23

López de Saro, Francisco, Roxana E. Georgescu, Frank Leu, and Mike O'Donnell. "Protein trafficking on sliding clamps." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1441 (January 29, 2004): 25–30. http://dx.doi.org/10.1098/rstb.2003.1361.

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The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase. Several other proteins and polymerases also interact with the clamp. These proteins bind the clamp at the same spot and use it in sequential fashion. First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase. At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase. This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp. The mechanism by which traffic flow on the β; clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition. It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.

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24

FİDAN, Özkan, and Somdutt MUJWAR. "In silico Evaluation of the Potential of Natural Products from Chili Pepper as Antiviral Agents Against Dna-Directed Rna Polymerase of the Monkeypox Virus." Bitlis Eren Üniversitesi Fen Bilimleri Dergisi 13, no. 1 (February 9, 2024): 277–91. http://dx.doi.org/10.17798/bitlisfen.1388403.

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This study focused on the discovery of new drug candidates effective against the monkeypox virus. Virtual screening was performed to evaluate the potential of chili pepper natural products against homology-modeled DNA-directed RNA polymerase of the monkeypox virus using molecular docking. Our findings revealed that structurally similar triterpenes such as α-amyrin, β-amyrin, and β-sitosterol had strong binding affinities towards the DNA-directed RNA polymerase and can inhibit this pivotal viral enzyme. The stability of one of the drug candidate molecules, α-amyrin with the strongest binding affinity towards the binding cavity of the enzyme was also confirmed via molecular dynamics simulation. This study showed that α-amyrin is a promising DNA-directed RNA polymerase inhibitor to treat monkeypox disease. It also paves the way for the idea of the potential dietary supplement candidate for monkeypox patients.

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25

Ma, Yi, Beilei Zhang, Meng Wang, Yanghui Ou, Jufang Wang, and Shan Li. "Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site." BioMed Research International 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/2906484.

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The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.

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Siau, Jia Wei, Samuel Nonis, Sharon Chee, Li Quan Koh, Fernando J. Ferrer, Christopher J. Brown, and Farid J. Ghadessy. "Directed co-evolution of interacting protein–peptide pairs by compartmentalized two-hybrid replication (C2HR)." Nucleic Acids Research 48, no. 22 (October 26, 2020): e128-e128. http://dx.doi.org/10.1093/nar/gkaa933.

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Abstract Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein–peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein–peptide pair. Escherichia coli cells co-expressing protein–peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein–peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher–SpyTag pair and co-selecting for functionally interacting variants.

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27

Santos, M. E., and J. W. Drake. "Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants." Genetics 138, no. 3 (November 1, 1994): 553–64. http://dx.doi.org/10.1093/genetics/138.3.553.

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Abstract Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity.

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Xia, G., L. Chen, T. Sera, M. Fa, P. G. Schultz, and F. E. Romesberg. "Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase." Proceedings of the National Academy of Sciences 99, no. 10 (May 14, 2002): 6597–602. http://dx.doi.org/10.1073/pnas.102577799.

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Yang, Han, Xiaoxin Ji, Xiao Li, Yunran Feng, Ke Zhang, Xuemin Guo, Zhixiong Zhong, and Xin Mu. "Colorimetry-Based Phosphate Measurement for Polymerase Elongation." BioMed Research International 2023 (January 23, 2023): 1–13. http://dx.doi.org/10.1155/2023/8296847.

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DNA detection, which includes the measurement of variants in sequences or the presence of certain genes, is widely used in research and clinical diagnosis. Both require DNA-dependent DNA polymerase-catalyzed strand extension. Currently, these techniques rely heavily on the instruments used to visualize the results. This study introduced a simple and direct colorimetric method to measure polymerase-directed elongation. First, pyrophosphate (PPi), a by-product of strand extension, is converted into phosphate (Pi). Phosphate levels were measured using either Mo-Sb or BIOMOL Green reagent. This study showed that this colorimetry can distinguish single-base variants and detect PCR products in preset stringent conditions, implicating the potential value of this strategy to detect DNA.

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Law, Julie A., Jiamu Du, Christopher J. Hale, Suhua Feng, Krzysztof Krajewski, Ana Marie S. Palanca, Brian D. Strahl, Dinshaw J. Patel, and Steven E. Jacobsen. "Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1." Nature 498, no. 7454 (May 1, 2013): 385–89. http://dx.doi.org/10.1038/nature12178.

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31

Bebenek, Kataryzna, and Thomas A. Kunkel. "The use of native T7 DNA polymerase for site-directed mutagenesis." Nucleic Acids Research 17, no. 13 (1989): 5408. http://dx.doi.org/10.1093/nar/17.13.5408.

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32

Guo, Wei, Shuhui Sun, Binghua Xie, Xiao-Jing Zhu, and Zhong-Min Dai. "T4 DNA polymerase improves the efficiency of multiple site-directed mutagenesis." Biotechnology & Biotechnological Equipment 30, no. 4 (April 12, 2016): 721–25. http://dx.doi.org/10.1080/13102818.2016.1170630.

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33

Fa, Ming, Annalisa Radeghieri, Allison A. Henry, and Floyd E. Romesberg. "Expanding the Substrate Repertoire of a DNA Polymerase by Directed Evolution." Journal of the American Chemical Society 126, no. 6 (February 2004): 1748–54. http://dx.doi.org/10.1021/ja038525p.

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34

Hadjimarcou, Michalis I., Robert J. Kokoska, Thomas D. Petes, and Linda J. Reha-Krantz. "Identification of a Mutant DNA Polymerase δ in Saccharomyces cerevisiae With an Antimutator Phenotype for Frameshift Mutations." Genetics 158, no. 1 (May 1, 2001): 177–86. http://dx.doi.org/10.1093/genetics/158.1.177.

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Abstract We propose that a β-turn-β structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol δ. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative β-turn-β structure. The mutant DNA pol δ has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol δ was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol δ in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

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Takami, K., S. Matsuda, A. Sono, and K. Sakaguchi. "A meiotic DNA polymerase from a mushroom, Agaricus bisporus." Biochemical Journal 299, no. 2 (April 15, 1994): 335–40. http://dx.doi.org/10.1042/bj2990335.

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A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2′,3′-dideoxythymidine 5′-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.

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Peramachi Palanivelu. "Polymerase and Proofreading Exonuclease Domains of the Nuclear-encoded DNA-dependent RNA Polymerase of Plant Mitochondria." World Journal of Advanced Research and Reviews 19, no. 2 (August 30, 2023): 989–1004. http://dx.doi.org/10.30574/wjarr.2023.19.2.1670.

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Mitochondria, found in all eukaryotic cells, play a crucial role in generating much needed biological energy for the cells in the form of adenosine triphosphates (ATPs). It is a semi-autonomous organelle and is partly controlled by its own genome and mostly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB which are essentially similar to the E. coli DNA polymerase I. The nuclear-encoded RNA polymerase (NEP) (EC 2.7.7.6) is imported from the nucleus and involves in the transcription of all mitochondrial genes. In Arabidopsis thaliana, the mitochondrial NEP showed 59.05% identity to the NEP of the chloroplasts, but only 28.24% identity to the T7 RNA polymerase, suggesting the NEPs of mitochondria and chloroplasts are distinctly different. However, in both the plant NEPs, the polymerase catalytic core and proofreading (PR) exonuclease domains are completely conserved. The mitochondrial NEP’s catalytic core from different plant sources is remarkably conserved and is in close agreement with other RNA polymerases reported already and possesses a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4, from the catalytic K. The catalytic metal-binding motifs are identified based on sequence similarity and site-directed mutagenesis (SDM) experiments. The PR exonuclease of the mitochondrial NEP belongs to the DEDD-superfamily of exonucleases.

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LOYA, Shoshana, Amira RUDI, Yoel KASHMAN, and Amnon HIZI. "Mode of inhibition of HIV reverse transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases." Biochemical Journal 324, no. 3 (June 15, 1997): 721–27. http://dx.doi.org/10.1042/bj3240721.

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A natural compound from the Red Sea sponge Ircinia sp., 2-hexaprenylhydroquinone (HPH), has been shown to be a general inhibitor of retroviral reverse transcriptases (from HIV-1, HIV-2 and murine leukaemia virus) as well as of cellular DNA polymerases (Escherichia coli DNA polymerase I, and DNA polymerases α and β). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-1 reverse transcriptase. HPH is a non-competitive inhibitor and binds the enzyme irreversibly with high affinity (Ki = 0.62 μM). The polar hydroxy groups have been shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased capacity to inhibit all DNA polymerases tested. Like the natural product, mHPH binds the enzyme independently at an allosteric site, but with reduced affinity (Ki = 7.4 μM). We show that HPH does not interfere with the first step of the polymerization process, i.e. the physical formation of the reverse-transcriptase–DNA complex. Consequently, we suggest that the natural inhibitor interferes with the subsequent steps of the overall reaction. Since HPH seems not to affect the affinity of dNTP for the enzyme (the Km is unchanged under conditions where the HPH concentration is increased), we speculate that its inhibitory capacity is derived from its effect on the nucleotidyl-transfer catalytic reaction. We suggest that such a mechanism of inhibition is typical of an inhibitor whose mode of inhibition should be common to all RNA- and DNA-directed polymerases.

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Zhong, Xuehua, Christopher J. Hale, Minh Nguyen, Israel Ausin, Martin Groth, Jonathan Hetzel, Ajay A. Vashisht, Ian R. Henderson, James A. Wohlschlegel, and Steven E. Jacobsen. "DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis." Proceedings of the National Academy of Sciences 112, no. 3 (January 5, 2015): 911–16. http://dx.doi.org/10.1073/pnas.1423603112.

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DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.

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Kurzynska-Kokorniak, Anna, Varuni K. Jamburuthugoda, Arkadiusz Bibillo, and Thomas H. Eickbush. "DNA-directed DNA Polymerase and Strand Displacement Activity of the Reverse Transcriptase Encoded by the R2 Retrotransposon." Journal of Molecular Biology 374, no. 2 (November 2007): 322–33. http://dx.doi.org/10.1016/j.jmb.2007.09.047.

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40

Ong, Jennifer L., David Loakes, Szymon Jaroslawski, Kathleen Too, and Philipp Holliger. "Directed Evolution of DNA Polymerase, RNA Polymerase and Reverse Transcriptase Activity in a Single Polypeptide." Journal of Molecular Biology 361, no. 3 (August 2006): 537–50. http://dx.doi.org/10.1016/j.jmb.2006.06.050.

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41

Wang, Yingying, Li-Ping Jiang, Shiwei Zhou, Sai Bi, and Jun-Jie Zhu. "DNA Polymerase-Directed Hairpin Assembly for Targeted Drug Delivery and Amplified Biosensing." ACS Applied Materials & Interfaces 8, no. 40 (September 30, 2016): 26532–40. http://dx.doi.org/10.1021/acsami.6b08597.

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42

Weiner, Michael P., Gina L. Costa, Warren Schoettlin, Janice Cline, Eric Mathur, and John C. Bauer. "Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction." Gene 151, no. 1-2 (December 1994): 119–23. http://dx.doi.org/10.1016/0378-1119(94)90641-6.

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Wendte, Jered M., Jeremy R. Haag, Olga M. Pontes, Jasleen Singh, Sara Metcalf, and Craig S. Pikaard. "The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation." Nucleic Acids Research 47, no. 17 (July 22, 2019): 9024–36. http://dx.doi.org/10.1093/nar/gkz615.

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Abstract In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

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Lelyveld, Victor S., Ziyuan Fang, and Jack W. Szostak. "Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity." Science 382, no. 6669 (October 27, 2023): 423–29. http://dx.doi.org/10.1126/science.adh5339.

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A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3′→P5′ phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post–transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.

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Rao, Venigalla B., and Nancy B. Saunders. "A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus." Gene 113, no. 1 (April 1992): 17–23. http://dx.doi.org/10.1016/0378-1119(92)90665-c.

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46

Mishra, Vibhor, Jasleen Singh, Feng Wang, Yixiang Zhang, Akihito Fukudome, Jonathan C. Trinidad, Yuichiro Takagi, and Craig S. Pikaard. "Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV." Proceedings of the National Academy of Sciences 118, no. 13 (March 22, 2021): e2019276118. http://dx.doi.org/10.1073/pnas.2019276118.

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In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV’s 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3′ ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

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Wrzesiński, Michał, Anetta Nowosielska, Jadwiga Nieminuszczy, and Elzbieta Grzesiuk. "Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells." Acta Biochimica Polonica 52, no. 1 (March 31, 2005): 139–47. http://dx.doi.org/10.18388/abp.2005_3499.

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Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.

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Chang, Seungwoo, Karel Naiman, Elizabeth S. Thrall, James E. Kath, Slobodan Jergic, Nicholas E. Dixon, Robert P. Fuchs, and Joseph J. Loparo. "A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes." Proceedings of the National Academy of Sciences 116, no. 51 (December 3, 2019): 25591–601. http://dx.doi.org/10.1073/pnas.1914485116.

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DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within theEscherichia colireplisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

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Eissenberg, J. C., R. Ayyagari, X. V. Gomes, and P. M. Burgers. "Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon." Molecular and Cellular Biology 17, no. 11 (November 1997): 6367–78. http://dx.doi.org/10.1128/mcb.17.11.6367.

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The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.

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Sigman, Meredith J., Kaushik Panda, Rachel Kirchner, Lauren L. McLain, Hayden Payne, John Reddy Peasari, Aman Y. Husbands, R. Keith Slotkin, and Andrea D. McCue. "An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation." Nature Plants 7, no. 11 (November 2021): 1461–74. http://dx.doi.org/10.1038/s41477-021-01008-7.

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AbstractIn mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

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Journal articles on the topic 'DNA-directed DNA polymerase'

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