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Journal articles on the topic 'And strand annealers'
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1
Sugiman-Marangos, Seiji N., Yoni M. Weiss, and Murray S. Junop. "Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB." Proceedings of the National Academy of Sciences 113, no. 16 (April 4, 2016): 4308–13. http://dx.doi.org/10.1073/pnas.1520847113.
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Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.
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Chalapati, Sachin, Conor A. Crosbie, Dixita Limbachiya, and Nimesh Pinnamaneni. "Direct oligonucleotide sequencing with nanopores." Open Research Europe 1 (August 24, 2021): 47. http://dx.doi.org/10.12688/openreseurope.13578.2.
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Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.
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Chalapati, Sachin, Conor A. Crosbie, Dixita Limbachiya, and Nimesh Pinnamaneni. "Direct oligonucleotide sequencing with nanopores." Open Research Europe 1 (May 12, 2021): 47. http://dx.doi.org/10.12688/openreseurope.13578.1.
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Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.
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Bartos, Jeremy D., Wensheng Wang, Jason E. Pike, and Robert A. Bambara. "Mechanisms by Which Bloom Protein Can Disrupt Recombination Intermediates of Okazaki Fragment Maturation." Journal of Biological Chemistry 281, no. 43 (August 31, 2006): 32227–39. http://dx.doi.org/10.1074/jbc.m606310200.
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Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures.
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Capitani, Gian Carlo, Jean-Claude Doukhan, Thomas Malcherek, and Michael Carpenter. "Strain modulation around inclusions in an annealed natural cordierite." European Journal of Mineralogy 13, no. 5 (September 27, 2001): 921–28. http://dx.doi.org/10.1127/0935-1221/2001/0013-0921.
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Bilang, R., A. Peterhans, A. Bogucki, and J. Paszkowski. "Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays." Molecular and Cellular Biology 12, no. 1 (January 1992): 329–36. http://dx.doi.org/10.1128/mcb.12.1.329.
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Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.
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Bilang, R., A. Peterhans, A. Bogucki, and J. Paszkowski. "Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays." Molecular and Cellular Biology 12, no. 1 (January 1992): 329–36. http://dx.doi.org/10.1128/mcb.12.1.329-336.1992.
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Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.
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Maryon, E., and D. Carroll. "Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination." Molecular and Cellular Biology 11, no. 6 (June 1991): 3278–87. http://dx.doi.org/10.1128/mcb.11.6.3278.
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Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.
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Maryon, E., and D. Carroll. "Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination." Molecular and Cellular Biology 11, no. 6 (June 1991): 3278–87. http://dx.doi.org/10.1128/mcb.11.6.3278-3287.1991.
Full textAbstract:
Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.
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Rauscher, G. "Sixtus-Tonks experiments on strand annealed 49Co-2V-Fe wires." IEEE Transactions on Magnetics 21, no. 5 (September 1985): 1930–32. http://dx.doi.org/10.1109/tmag.1985.1064075.
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Zhu, Yali, Zetang Wu, M. Cristina Cardoso, and Deborah S. Parris. "Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase." Journal of Virology 84, no. 15 (May 5, 2010): 7459–72. http://dx.doi.org/10.1128/jvi.01875-09.
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ABSTRACT The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo−) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo− HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo− HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.
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Davis, Allison P., and Lorraine S. Symington. "The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing." Genetics 159, no. 2 (October 1, 2001): 515–25. http://dx.doi.org/10.1093/genetics/159.2.515.
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Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.
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Wang, Zhenghui, Ke Zhang, Karen L. Wooley, and John-Stephen Taylor. "Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated Probes." Journal of Nucleic Acids 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/962652.
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Probes for monitoring mRNA expressionin vivoare of great interest for the study of biological and biomedical problems, but progress has been hampered by poor signal to noise and effective means for delivering the probes into live cells. Herein we report a PNA·DNA strand displacement-activated fluorescent probe that can image the expression of iNOS (inducible nitric oxide synthase) mRNA, a marker of inflammation. The probe consists of a fluorescein labeled antisense PNA annealed to a shorterDABCYLplus-labeled DNA which quenches the fluorescence, but when the quencher strand is displaced by the target mRNA the fluorescence is restored. DNA was used for the quencher strand to facilitate electrostatic binding of the otherwise netural PNA strand to a cationic shell crosslinked knedel-like (cSCK) nanoparticle which can deliver the PNA·DNA duplex probe into cells with less toxicity and greater efficiency than other transfection agents. RAW 264.7 mouse macrophage cells transfected with the iNOS PNA·DNA probe via the cSCK showed a 16 to 54-fold increase in average fluorescence per cell upon iNOS stimulation. The increase was 4 to 7-fold higher than that for a non-complementary probe, thereby validating the ability of a PNA·DNA strand displacement-activated probe to image mRNA expressionin vivo.
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Caldwell, Brian J., Andrew Norris, Ekaterina Zakharova, Christopher E. Smith, Carter T. Wheat, Deepanshu Choudhary, Marcos Sotomayor, Vicki H. Wysocki, and Charles E. Bell. "Oligomeric complexes formed by Redβ single strand annealing protein in its different DNA bound states." Nucleic Acids Research 49, no. 6 (March 8, 2021): 3441–60. http://dx.doi.org/10.1093/nar/gkab125.
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Abstract Redβ is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redβ forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redβ in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redβ forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redβ in cells active for recombination and find it to range from 7 to 27 μM. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.
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Driscoll, Mark D., Ganesan Sathya, Layla F. Saidi, Michael S. DeMott, Russell Hilf, and Robert A. Bambara. "An Explanation for Observed Estrogen Receptor Binding to Single-Stranded Estrogen-Responsive Element DNA." Molecular Endocrinology 13, no. 6 (June 1, 1999): 958–68. http://dx.doi.org/10.1210/mend.13.6.0296.
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Abstract Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.
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Wu, Tiyun, Jianhui Guo, Julian Bess, Louis E. Henderson, and Judith G. Levin. "Molecular Requirements for Human Immunodeficiency Virus Type 1 Plus-Strand Transfer: Analysis in Reconstituted and Endogenous Reverse Transcription Systems." Journal of Virology 73, no. 6 (June 1, 1999): 4794–805. http://dx.doi.org/10.1128/jvi.73.6.4794-4805.1999.
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ABSTRACT We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3 Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3′ end of tRNA3 Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3′ rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3′-terminal bases by RNase H (added either in cis ortrans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.
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Li, Jinbao, Huize Sun, Yulin Huang, Yali Wang, Yuyan Liu, and Xuefeng Chen. "Pathways and assays for DNA double-strand break repair by homologous recombination." Acta Biochimica et Biophysica Sinica 51, no. 9 (July 10, 2019): 879–89. http://dx.doi.org/10.1093/abbs/gmz076.
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AbstractDouble strand breaks (DSBs) are the most detrimental type of DNA damage that must be repaired to ensure genome integrity and cell survival. Unrepaired or improperly repaired DSBs can potentially cause tumorigenesis or cell death. DSBs are primarily repaired by non-homologous end joining or homologous recombination (HR). The HR pathway is initiated by processing of the 5′-end of DSBs to generate 3′-end single-strand DNA (ssDNA). Furthermore, the intermediate is channeled to one of the HR sub-pathways, including: (i) double Holliday junction (dHJ) pathway, (ii) synthesis-dependent strand annealing (SDSA), (iii) break-induced replication (BIR), and (iv) single-strand annealing (SSA). In the dHJ sub-pathway, the 3′-ssDNA coated with Rad51 recombinase performs homology search and strand invasion, forming a displacement loop (D-loop). Capture of the second end by the D-loop generates a dHJ intermediate that is subsequently dissolved by DNA helicase or resolved by nucleases, producing non-crossover or crossover products. In SDSA, the newly synthesized strand is displaced from the D-loop and anneals to the end on the other side of the DSBs, producing non-crossovers. In contrast, BIR repairs one-end DSBs by copying the sequence up to the end of the template chromosome, resulting in translocation or loss of heterozygosity. SSA takes place when resection reveals flanking homologous repeats that can anneal, leading to deletion of the intervening sequences. A variety of reporter assays have been developed to monitor distinct HR sub-pathways in both Saccharomyces cerevisiae and mammals. Here, we summarize the principles and representative assays for different HR sub-pathways with an emphasis on the studies in the budding yeast.
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Annex, B. H., and R. S. Williams. "Mitochondrial DNA structure and expression in specialized subtypes of mammalian striated muscle." Molecular and Cellular Biology 10, no. 11 (November 1990): 5671–78. http://dx.doi.org/10.1128/mcb.10.11.5671.
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Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.
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Annex, B. H., and R. S. Williams. "Mitochondrial DNA structure and expression in specialized subtypes of mammalian striated muscle." Molecular and Cellular Biology 10, no. 11 (November 1990): 5671–78. http://dx.doi.org/10.1128/mcb.10.11.5671-5678.1990.
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Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.
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Sung, Wing L., Diana M. Zahab, Fei-Long Yao, and Cherk S. Tam. "Hybrid gene synthesis: its application to the assembly of DNA sequences encoding the human parathyroid hormones and analogues." Biochemistry and Cell Biology 64, no. 2 (February 1, 1986): 133–38. http://dx.doi.org/10.1139/o86-021.
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Bypassing any intermediate steps of purification and gene assembly, several synthetic oligonucleotides constituting a DNA duplex with a small base-mismatching region were phosphorylated, annealed, and ligated directly into a linearized plasmid vector. After transformation in bacteria, the two plasmid strands individualy yielded two different plasmids bearing altered versions of the same gene. Via this approach, DNA coding sequences of the human parathyroid hormone and analogues were synthesized and cloned in Escherichia coli.
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Ma, Jia-Lin, Eun Mi Kim, James E. Haber, and Sang Eun Lee. "Yeast Mre11 and Rad1 Proteins Define a Ku-Independent Mechanism To Repair Double-Strand Breaks Lacking Overlapping End Sequences." Molecular and Cellular Biology 23, no. 23 (December 1, 2003): 8820–28. http://dx.doi.org/10.1128/mcb.23.23.8820-8828.2003.
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ABSTRACT End joining of double-strand breaks (DSBs) requires Ku proteins and frequently involves base pairing between complementary terminal sequences. To define the role of terminal base pairing in end joining, two oppositely oriented HO endonuclease cleavage sites separated by 2.0 kb were integrated into yeast chromosome III, where constitutive expression of HO endonuclease creates two simultaneous DSBs with no complementary end sequence. Lack of complementary sequence in their 3′ single-strand overhangs facilitates efficient repair events distinctly different from when the 3′ ends have a 4-bp sequence base paired in various ways to create 2- to 3-bp insertions. Repair of noncomplementary ends results in a set of nonrandom deletions of up to 302 bp, annealed by imperfect microhomology of about 8 to 10 bp at the junctions. This microhomology-mediated end joining (MMEJ) is Ku independent, but strongly dependent on Mre11, Rad50, and Rad1 proteins and partially dependent on Dnl4 protein. The MMEJ also occurs when Rad52 is absent, but the extent of deletions becomes more limited. The increased gamma ray sensitivity of rad1Δ rad52Δ yku70Δ strains compared to rad52Δ yku70Δ strains suggests that MMEJ also contributes to the repair of DSBs induced by ionizing radiation.
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Daley, James M., and Thomas E. Wilson. "Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length." Molecular and Cellular Biology 25, no. 3 (February 1, 2005): 896–906. http://dx.doi.org/10.1128/mcb.25.3.896-906.2005.
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ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.
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Locatelli, Giada A., Helmut Pospiech, Nicolas Tanguy Le Gac, Barbara van Loon, Ulrich Hubscher, Sinikka Parkkinen, Juhani E. Syväoja, and Giuseppe Villani. "Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase ϵ in the presence of replication protein A and proliferating-cell nuclear antigen." Biochemical Journal 429, no. 3 (July 14, 2010): 573–82. http://dx.doi.org/10.1042/bj20100405.
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DNA pol (polymerase) ϵ is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol ϵ can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol ϵ. RPA inhibited the elongation by human DNA pol ϵ on templates annealed to short primers. PCNA did not influence the elongation by DNA pol ϵ and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol ϵ after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol ϵ to replicate past the AP site, independent of the primer length.
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Andersen, Bjørn, Nicola Margiotta, Mauro Coluccia, Giovanni Natile, and Einar Sletten. "Antitumor Trans Platinum DNA Adducts: NMR and HPLC Study of the Interaction Between a trans-Pt Iminoether Complex and the Deoxy Decamer d(CCTCGCTCTC)·d(GAGAGCGAGG)." Metal-Based Drugs 7, no. 1 (January 1, 2000): 23–32. http://dx.doi.org/10.1155/mbd.2000.23.
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The single-stranded oligonucleotide 5′-d(CCTCGCTCTC) (I) was reacted with the antitumor trans platinum iminoderivative trans-[PtCl2{E-HN = C(OMe)Me}2] (trans-EE) and subsequently annealed with its complementary strand 5′-d(GAGAGCGAGG) (II). The platinated duplex was characterized by 1D and 2D proton NMR spectroscopy at 600 MHz. In agreement with previous studies by different techniques trans-EE was found to form a monofunctional adduct with the duplex involving the guanine residue. The modification by trans-EE has been found to induce only minor local distortion in the duplex geometry. Two key crosspeaks observed in the NOESY map corresponding to a close contact between G5-H8 and the methoxy and the methyl group, respectively, enabled us to dock the trans-EE complex with the duplex by geometry optimization. The results support the idea that the antitumor activity of trans-EE is related to lesion of DNA fundamentally different from that of cisplatin. Unexpectedly, the NOESY spectra indicated that at the high NaCl concentration used (0.2 M) the duplex was found to undergo slow deplatination. This was subsequently proved by HPLC. In a separate experiment on platination of the single strand in a salt free environment the HPLC analysis showed that the monofunctional adduct was not deplatinated, however, after 24 hours, additidnal minor isomers were detected.
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Kumar, Pawan. "Non-intertwined strands of plasmid DNA contradict the Watson and Crick model of DNA structure." F1000Research 8 (November 10, 2020): 356. http://dx.doi.org/10.12688/f1000research.18134.3.
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According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of 0.5 N NaOH to pUC19 or pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when subjected to renaturing conditions, HmDNA from NaOH-denatured pUC19 re-annealed to form native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli. These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.
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Ross, Jason P., Isao Suetake, Shoji Tajima, and Peter L. Molloy. "Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA–DNA heteroduplex substrates." Biochemical Journal 432, no. 2 (November 12, 2010): 323–32. http://dx.doi.org/10.1042/bj20100579.
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The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA–DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA–DNA heteroduplex context and whether a RNA–DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA–DNA heteroduplexes, DNA bubbles and R-loops. Dnmt methylation activity on the model substrates was quantified with initial velocity assays, novel ARORA (annealed RNA and DNA oligonucleotide-based methylation-sensitive restriction enzyme analysis), tBS (tagged-bisulfite sequencing) and the quantitative PCR-based method MethylQuant. We found that RNA–DNA heteroduplexes and R-loops are poor substrates for methylation by both the maintenance (Dnmt1) and de novo (Dnmt3a and Dnmt3b) Dnmts. These results suggest the proposed RNA/DNA model of TGS in mammals is unlikely. Analysis of tagged-bisulfite genomic sequencing led to the unexpected observation that Dnmt1-dN can methylate cytosines in a non-CpG context in DNA bubbles. This may have relevance in DNA replication and silencing of transcriptionally active loci in vivo.
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Driscoll, Mark D., Marie-Pierre Golinelli, and Stephen H. Hughes. "In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing." Journal of Virology 75, no. 2 (January 15, 2001): 672–86. http://dx.doi.org/10.1128/jvi.75.2.672-686.2001.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA3 Lys primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5′ end of the RNA that remained annealed to the 3′ end of the minus-strand strong-stop DNA only if NC was present in the reaction.
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Lay, Marla J., and Carl T. Wittwer. "Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR." Clinical Chemistry 43, no. 12 (December 1, 1997): 2262–67. http://dx.doi.org/10.1093/clinchem/43.12.2262.
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Abstract A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5TM in the presence of a 3′-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 °C; if the single-base mutation is present, the probe melts at 57 °C. Concurrent amplification and analysis from genomic DNA takes 20–45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
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Driscoll, Mark D., and Stephen H. Hughes. "Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences." Journal of Virology 74, no. 19 (October 1, 2000): 8785–92. http://dx.doi.org/10.1128/jvi.74.19.8785-8792.2000.
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ABSTRACT Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (−sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with −sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic −sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3′ end of the −sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.
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Levin, H. L. "A novel mechanism of self-primed reverse transcription defines a new family of retroelements." Molecular and Cellular Biology 15, no. 6 (June 1995): 3310–17. http://dx.doi.org/10.1128/mcb.15.6.3310.
Full textAbstract:
Retroviruses and long terminal repeat (LTR)-containing retrotransposons initiate reverse transcription by using a specific tRNA primer than anneals to the primer-binding site of the retroelement transcript. Sequences from a large number of retroviruses and LTR-containing retrotransposons had indicated that the role of tRNAs in priming reverse transcription is universal among these LTR-containing retroelements. Data presented here strongly support the surprising conclusion that Tf1, a highly active LTR-containing retrotransposon isolated from Schizosaccharomyces pombe, undergoes a novel self-priming process that requires hybridization between the primer-binding site and the first 11 bases of the Tf1 transcript. Single-base mutations in these regions block transposition and reverse transcription, while compensatory mutations that reestablish complementarily rescue both defects. In addition, the sequence of the minus-strand RNA primer of reverse transcription was consistent with its being derived from the 5' end of the Tf1 transcript. Evidence that this mechanism defines a new family of retroelements is presented.
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Varkey, Jacob, Mark Hannibal, John Brantley, Brian Stewart, and Michael Beer. "Gene-Specific Labeling For An Electron Microscopic Study of Chromosomal Proteins." Proceedings, annual meeting, Electron Microscopy Society of America 43 (August 1985): 720–21. http://dx.doi.org/10.1017/s0424820100120291.
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With the aim of understanding the control of gene expression we are developing techniques to study the proteins bound to particular genes at various stages of activity. To accomplish this we have worked out a gene specific labeling procedure and are testing it with the 5S RNA genes of Xenopus.Our approach is based on the gene specific purification proposed by Workman and Langmore (1984). Chromatin is digested with a suitable restriction enzyme followed by lambda exonuclease to yield a protruding 3’ end. DMA from the same gene is cut with the same restriction enzyme and nick translated with biotin-dUTP. Then it is digested with exonuclease III to produce a protruding single strand complementary to that of the chromatin. Probe and chromatin are annealed to each other under gentle conditions (0. 1m NaCl; 37°C), Avidin linked to Ferritin is added to decorate the biotin containing regions of the probe and the material is examined in the electron microscope. The chromatin pieces of interest can be recognized by the attached Ferritin labeled probe.
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Rong, Liwei, Chen Liang, Mayla Hsu, Xiaofeng Guo, Bernard P. Roques, and Mark A. Wainberg. "HIV-1 Nucleocapsid Protein and the Secondary Structure of the Binary Complex Formed between tRNALys.3and Viral RNA Template Play Different Roles during Initiation of (−) Strand DNA Reverse Transcription." Journal of Biological Chemistry 276, no. 50 (October 15, 2001): 47725–32. http://dx.doi.org/10.1074/jbc.m105124200.
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In human immunodeficiency virus type 1 (HIV-1), the tRNALys.3primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions. Initiation of reverse transcription from this complex has been shown to be distinguished from subsequent elongation by early pausing events, such as at the +1 and +3 nucleotide positions. One major concern regarding the biological relevance of these results is that most kinetic studies of HIV-1 reverse transcription have been performed using tRNALys.3-viral (v) RNA complexes that were formed by heat annealing. In contrast, tRNALys.3in viruses is placed onto the primer binding site by nucleocapsid (NC) sequences of the Gag protein. In this study, we have further characterized the initiation features of reverse transcription in the presence of HIV-1 NC protein. In contrast to results obtained with a heat-annealed tRNALys.3·vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the +1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was actually still present during reverse transcription. This activity of NC required both zinc finger motifs, as demonstrated by experiments that employed zinc finger-mutated forms of NC protein (H23C NC and ddNC), supporting the involvement of the zinc fingers in the RNA chaperone activity of NC. However, NC was not able to help reverse transcriptase to escape the +3 pausing event. Mutagenesis of a stem structure within the tRNALys.3. vRNA complex led to disappearance of the +3 pausing event as well as to significantly reduced rates of reverse transcription. Thus, this stem structure is essential for optimal reverse transcription, despite its role in promotion of the +3 pausing event.
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Jalanko, Anu, Juha Kere, Erkki Savilahti, Marianne Schwartz, Ann-Christine Syvänen, Marjut Ranki, and Hans Söderlund. "Screening for Defined Cystic Fibrosis Mutations by Solid-Phase Minisequencing." Clinical Chemistry 38, no. 1 (January 1, 1992): 39–43. http://dx.doi.org/10.1093/clinchem/38.1.39.
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Abstract We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.
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Szczepanska, Agnieszka, Marta Wojnicka, and Anna Kurzynska-Kokorniak. "The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer." International Journal of Molecular Sciences 22, no. 16 (August 13, 2021): 8690. http://dx.doi.org/10.3390/ijms22168690.
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Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.
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Tsurumi, T. "Primer terminus recognition and highly processive replication by Epstein-Barr virus DNA polymerase." Biochemical Journal 280, no. 3 (December 15, 1991): 703–8. http://dx.doi.org/10.1042/bj2800703.
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The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle. It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template. Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself. ATP was not required either for primer terminus recognition or for sustainment of polymerization. The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized. These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase. The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation. The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule. The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication.
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Thakar, Tanay, and George-Lucian Moldovan. "The emerging determinants of replication fork stability." Nucleic Acids Research 49, no. 13 (May 12, 2021): 7224–38. http://dx.doi.org/10.1093/nar/gkab344.
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Abstract A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy.
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Cen, Shan, Yue Huang, Ahmad Khorchid, Jean-Luc Darlix, Mark A. Wainberg, and Lawrence Kleiman. "The Role of Pr55gag in the Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Genomic RNA." Journal of Virology 73, no. 5 (May 1, 1999): 4485–88. http://dx.doi.org/10.1128/jvi.73.5.4485-4488.1999.
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ABSTRACT During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3 Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3 Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3 Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55 gag or Pr160 gag-pol . In vivo placement of tRNA3 Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55 gag or mutant Pr160 gag-pol , and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55 gag inhibit tRNA3 Lysplacement. The NC mutations in Pr55 gag reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3 Lys even in the presence of wild-type Pr55 gag . This dominant phenotype may indicate that the mutant Pr55 gag is disrupting an ordered Pr55 gag structure responsible for the annealing of tRNA3 Lys to genomic RNA.
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Wardhani, Bantari W. K., Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, and Mitsuyasu Kato. "TMEPAI genome editing in triple negative breast cancer cells." Medical Journal of Indonesia 26, no. 1 (May 16, 2017): 14–8. http://dx.doi.org/10.13181/mji.v26i1.1871.
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Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.
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Pinz, Kevin G., and Daniel F. Bogenhagen. "Efficient Repair of Abasic Sites in DNA by Mitochondrial Enzymes." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1257–65. http://dx.doi.org/10.1128/mcb.18.3.1257.
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ABSTRACT Mutations in mitochondrial DNA (mtDNA) cause a variety of relatively rare human diseases and may contribute to the pathogenesis of other, more common degenerative diseases. This stimulates interest in the capacity of mitochondria to repair damage to mtDNA. Several recent studies have shown that some types of damage to mtDNA may be repaired, particularly if the lesions can be processed through a base excision mechanism that employs an abasic site as a common intermediate. In this paper, we demonstrate that a combination of enzymes purified from Xenopus laevis mitochondria efficiently repairs abasic sites in DNA. This repair pathway employs a mitochondrial class II apurinic/apyrimidinic (AP) endonuclease to cleave the DNA backbone on the 5′ side of an abasic site. A deoxyribophosphodiesterase acts to remove the 5′ sugar-phosphate residue left by AP endonuclease. mtDNA polymerase γ fills the resulting 1-nucleotide gap. The remaining nick is sealed by an mtDNA ligase. We report the first extensive purification of mtDNA ligase as a 100-kDa enzyme that functions with an enzyme-adenylate intermediate and is capable of ligating oligo(dT) strands annealed to poly(rA). These properties together with preliminary immunological evidence suggest that mtDNA may be related to nuclear DNA ligase III.
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Miglietta, Giulia, Marco Russo, and Giovanni Capranico. "G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders." Nucleic Acids Research 48, no. 21 (November 2, 2020): 11942–57. http://dx.doi.org/10.1093/nar/gkaa944.
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Abstract Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.
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Earnshaw, David L., and Andrew J. Pope. "FlashPlate Scintillation Proximity Assays for Characterization and Screening of DNA Polymerase, Primase, and Helicase Activities." Journal of Biomolecular Screening 6, no. 1 (February 2001): 39–46. http://dx.doi.org/10.1177/108705710100600106.
Full textAbstract:
DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gelbased techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [3H]-dNTPs onto the growing primer 3′ end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [3H]-rNTPs or by subsequent polymerase extension with [3H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [33P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.
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Pule, Martin A., Alexandra Rousseau, Elio F. Vanin, Malcolm K. Brenner, and Helen E. Heslop. "Flanking-Sequence Exponential Anchored (FLEA) PCR - a Sensitive and Highly Specific Method for Detecting Retroviral Integrant-Host-Junction Sequences." Blood 104, no. 11 (November 16, 2004): 2112. http://dx.doi.org/10.1182/blood.v104.11.2112.2112.
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Abstract Retroviral vectors integrate in the host cell genome and are widely used for gene therapy. Since integration is largely random, each site represents a unique integration event and can be used to study clonal evolution of engrafted cells. Integration can however be deleterious, and has been associated with oncogenesis. To date, PCR methods for studying the integrant-host junction (IHJ) have been laborious, unspecific and difficult to perform on complex samples. We have developed a method that is simpler and highly specific. We generate libraries of integrant-host junctions which can then be resolved by sequencing. The approach works even for analyses of highly complex samples. 1) Linear PCR is performed on sample DNA with biotinylated primer annealing to the 5′ end of the LTR. The subsequent longer biotinylated linear PCR fragments which contain IHJ or internal junctions are separated from genomic DNA with streptavidin paramagnetic beads. 2) We add an anchor primer with a known 5′ sequence and several degenerate 3′ bases. 3) A complimentary strand from this primer to the LTR is generated with T7 DNA polymerase. 4) A blocking oligo is added to the polymerase mixture, which anneals just before the 3′LTR of the internal junction and prevents the complementary strand from the internal sequence being generated, so that >90% of the anchored DNA contains external flanking genomic sequence. 5) We PCR amplify the material from a nested primer in the LTR to a primer, which anneals to the known sequence of the anchor primer. 6) Finally, a smear is generated which contains randomly anchored IHJs. FLEA-PCR was designed so that the most 5′ 33bp of the LTR (which contain an NheI site) are unprimed allowing easy validation of PCR product. Sequencing of these smears reveals that >99% of these DNA fragments contain this 33bp of 5′ extremity and are preceded by genomic sequences. With blocking, 90% of sequences are external IHJs. To validate the technique, 6 HeLa cell clones containing a single integrant were studied. The 5′ IHJ was characterized by FLEA-PCR amplification. The 3′ IHJ was successfully amplified by PCR with a primer designed on prediction from the human genome sequence and the 4bp repeat of junctional sequences was evident in all HeLa clones. Clonable smears can be generated with samples containing <0.1% transduced cells. The mean length of flanking genomic sequence is 97bp (ranging from 0 to 674bp). Digestion of smears amplified with a 6FAM-labelled LTR primer with CviJI and capillary electropheresis analysis allows precise counting of sample complexity. More accurately, random sequencing of smear can be assembled by custom written software. It is possible to study samples with over 80 different integrants in a single experiment. There are several advantages of using FLEA-PCR over restriction digest based methods. There is less PCR artifact. Since integrants yield varying fragments of random length, there is no PCR bias caused by differing amplicon lengths. This is of particular benefit when a short internal band can "swamp" the reaction. Secondly, no bias is accrued due to the frequency of occurrence of a particular restriction site. Finally, the technique is highly compatible with automation and high-throughput sequencing. This approach should facilitate IHJ analysis for assessing both retroviral safety and the biologic fate of transduced cells.
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Wityk, Paweł, Rafał Piątek, Robert Nowak, and Dorota Kostrzewa-Nowak. "Generation and Characterization of a DNA-GCN4 Oligonucleotide-Peptide Conjugate: The Impact DNA/Protein Interactions on the Sensitization of DNA." Molecules 25, no. 16 (August 10, 2020): 3630. http://dx.doi.org/10.3390/molecules25163630.
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Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.
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Gauthier, Florian, Jean-Rémi Bertrand, Jean-Jacques Vasseur, Christelle Dupouy, and Françoise Debart. "Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers." Molecules 25, no. 11 (June 11, 2020): 2714. http://dx.doi.org/10.3390/molecules25112714.
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Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.
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Kaczmarek, Frank S., Richard P. Zaniewski, Thomas D. Gootz, Dennis E. Danley, Mahmoud N. Mansour, Matt Griffor, Ajith V. Kamath, et al. "Cloning and Functional Characterization of an NAD+-Dependent DNA Ligase from Staphylococcus aureus." Journal of Bacteriology 183, no. 10 (May 15, 2001): 3016–24. http://dx.doi.org/10.1128/jb.183.10.3016-3024.2001.
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ABSTRACT A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD+-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD+-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD+-dependent DNA ligase fromB. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.
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Inagaki, Takayuki, Shoki Asahi, Kenji Ogawa, Taku Nakagawa, Toshiaki Nikai, Kiyofumi Yamada, Tetsuya Yagi, and Kei-ichi Uchiya. "1654. Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium using the Amplification Refractory Mutation System-Loop-Mediated Isothermal Amplification method." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S815—S816. http://dx.doi.org/10.1093/ofid/ofaa439.1832.
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Abstract Background Clarithromycin (CLR) is the key drug in multidrug therapy for Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility is correlated with a clinical response in these diseases. In the case of CLR-resistant MAC, a point mutation is present at either position 2058 or 2059 of the peptidyl transferase active center in the domain V region of 23S rRNA at the macrolide binding site. Using conventional investigation, we clarified the correlation between drug susceptibility testing and mutation of drug resistance genes. In this study, we adapted a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene in M. avium isolates (Figure 1). Furthermore, we evaluated the usefulness as point-of-care testing (POCT) technology using clinical isolates. Figure 1. The designs of CLR resistance A2058G mutant-type mismatch primers used for the ARMS-LAMP assay. a) A strand-displacing DNA polymerase extends the DNA from FIP while separating from the DNA chain. The primer F3 binds to its complementary region on the DNA to displace the newly synthesized DNA. An analogous reaction is performed by BIP and B3. α (α = A, wild type; G, A2058G) and β (β = A, wild type; C, A2058G) are indicated by the point mutation at position 2058 of the 23S rRNA gene. The bold area indicates the mismatched base C (cytosine). b) The synthesized DNA self-anneals because of the complementary region at both ends and forms ‘dumbbell’ structures. c) After repeated rounds, a complementary region on the same chain is amplified. Methods Primers for ARMS-LAMP were designed using PrimerExplorerV5 software based on the nucleotide sequence data for 23S rRNA in M. avium strain 104 (Figure 2). Using the minimum inhibitory concentration of CLR, drug susceptibility was determined for 18 clinical M. avium isolates. Of these, eight CLR-susceptible and 10 CLR-resistant strains were analyzed by sequencing the 23S rRNA gene and ARMS-LAMP. Figure 2. Alignment of the nucleotide sequences including the domain V region of 23S rRNA at the macrolide binding site. The constructed LAMP primer sets are shown in solid boxes (forward primers, F1-3) and dashed boxes (backward primers, B1-3). The bold area indicates the point mutation at position 2058 or 2059 of the 23S rRNA gene. Results Sequence analysis revealed that all eight CLR-sensitive strains tested were wild type, whereas all 10 CLR-resistant strains were mutants. Using ARMS-LAMP, no amplification with the mutant-type mismatch primer sets (MTPS) was observed in the eight wild-type strains, but amplification was observed with MTPS in the 10 mutant strains (Table 1). Table 1. MICs of CLR and results of ARMS–LAMP using Mycobacterium avium isolates. Conclusion The developed rapid detection method for the CLR resistance gene using ARMS-LAMP can determine drug resistance in a few hours without the need for special equipment. ARMS-LAMP may be a new clinically beneficial POCT technology for examination that is novel and extremely practical. Disclosures All Authors: No reported disclosures
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"Significance of strand configuration in self-replicating RNA molecules." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 350, no. 1334 (December 29, 1995): 345–52. http://dx.doi.org/10.1098/rstb.1995.0169.
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The kinetic theory of replication has been extended to include dual mechanisms for conversion of self-annealed single-strand RNA to double-strand molecules, which do not replicate. An analysis of experimental results established that the replicate-template annealing reaction during transcription significantly retarded replication in vitro among three RNA variants copied by Qβ replicase. Annealing between complementary RNA strands free in solution had far less significance. The finding that an RNA variant can be replicated in a multiple hairpin configuration, but not as its single, long hairpin conformer, the correlation between stability of strand secondary structure and replicative fitness, and a lack of homology in the internal sequence of RNA variants copied by Qβ replicase support the conclusion that template competence depends on strand configuration, independent of most of the underlying base sequence. Occurrence of self-annealed strands in the Qβ replicase system was attributed to its reliance on RNA-driven strand separation, in the absence of enzyme catalysed strand unwinding. A ‘configuration before sequence’ path to self-replication exhibited a substantially lower combinatorial barrier than standard sequence-dependent evolution. RNA-dependent RNA synthesis in the Qβ system thus displays features of an RNA World and, interestingly, they reveal a rapid path for evolution of the first self-replicating molecule on Earth.
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Jahns, Hartmut, Rohan Degaonkar, Peter Podbevsek, Swati Gupta, Anna Bisbe, Krishna Aluri, John Szeto, et al. "Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing." Nucleic Acids Research, September 11, 2021. http://dx.doi.org/10.1093/nar/gkab724.
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Abstract In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.
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Li, Feng, Xavier Badel, Jan Linnros, and John B. Wiley. "Colloidal Crystal Wires from Directed Assembly." MRS Proceedings 872 (2005). http://dx.doi.org/10.1557/proc-872-j20.3.
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AbstractColloidal crystal wires with tubular-like packings are prepared by the directed assembly of spheres into cylindrical one-dimensional channels. Silica spheres are infiltrated into porous silicon membranes, treated with silane, and annealed. Single annealing cycles are found to result in colloidal crystal wires with varied packing geometries, while repeated annealing produces a thin translucent silica sheath around the wires. Packing in the wires varies with the relative channel diameter of the silicon membrane where typical wires contain 4 to 7 helical strands. Both chiral and achiral packing geometries are observed. The fabrication of these wires is discussed and the relationship between channel size and packing structure detailed.