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BiopolymersStudies of catalytically active DNA sequences have expanded considerably since the first artificial deoxyribozyme was identified in 1994. Nevertheless, the field is... (Review)
Review
Studies of catalytically active DNA sequences have expanded considerably since the first artificial deoxyribozyme was identified in 1994. Nevertheless, the field is still quite young, and advances in both fundamental understanding and practical applications of deoxyribozymes are still developing. Deoxyribozymes that either cleave or ligate two RNA substrates have been most widely investigated, and this review describes recent advances in the fundamental studies and applications of these DNA enzymes. Deoxyribozymes with catalytic activities other than RNA ligation and cleavage are also increasingly pursued, and this review covers several key examples.
Topics: Catalysis; DNA, Catalytic; Molecular Structure; RNA; Ultraviolet Rays
PubMed: 17647280
DOI: 10.1002/bip.20813 -
The Journal of Biological Chemistry Feb 2013DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA...
DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). ROS1 and its homologs remove 5-methylcytosine and incise the sugar backbone at the abasic site, thus initiating a base excision repair pathway that finally inserts an unmethylated cytosine. The DNA 3'-phosphatase ZDP processes some of the incision products generated by ROS1, allowing subsequent DNA polymerization and ligation steps. In this work, we examined the possible role of plant XRCC1 (x-ray cross-complementing group protein 1) in DNA demethylation. We found that XRCC1 interacts in vitro with ROS1 and ZDP and stimulates the enzymatic activity of both proteins. Furthermore, extracts from xrcc1 mutant plants exhibit a reduced capacity to complete DNA demethylation initiated by ROS1. An anti-XRCC1 antibody inhibits removal of the blocking 3'-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis.
Topics: 5-Methylcytosine; Arabidopsis; Arabidopsis Proteins; DNA; DNA Glycosylases; DNA Methylation; DNA Repair; DNA-Binding Proteins; Epigenesis, Genetic; Gene Silencing; Models, Biological; Models, Genetic; Mutation; Nuclear Proteins; Protein Binding; Reactive Oxygen Species; X-ray Repair Cross Complementing Protein 1
PubMed: 23316050
DOI: 10.1074/jbc.M112.427617 -
Virology Jul 1996Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with...
Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.
Topics: Adenosine Triphosphate; DNA; DNA Ligases; DNA, Single-Stranded; Escherichia coli; Kinetics; Oligodeoxyribonucleotides; Recombinant Proteins; Vaccinia virus
PubMed: 8661420
DOI: 10.1006/viro.1996.0358 -
F1000Research 2019The relationship between varicoceles and subfertility is well-established, but recent evidence suggests that varicoceles may cause global testicular dysfunction. This... (Review)
Review
The relationship between varicoceles and subfertility is well-established, but recent evidence suggests that varicoceles may cause global testicular dysfunction. This has led to exploration into expanding the indications for varicocelectomy. This review examines the literature regarding varix ligation as a treatment for non-obstructive azoospermia, elevated DNA fragmentation, and hypogonadism.
Topics: Azoospermia; DNA Fragmentation; Humans; Hypogonadism; Ligation; Male; Testis; Varicocele
PubMed: 31543949
DOI: 10.12688/f1000research.19579.1 -
Biochemical and Biophysical Research... Mar 2002We previously reported that HMGB1, which originally binds to chromatin in a manner competitive with linker histone H1 to modulate chromatin structure, enhances both...
We previously reported that HMGB1, which originally binds to chromatin in a manner competitive with linker histone H1 to modulate chromatin structure, enhances both intra-molecular and inter-molecular ligations. In this paper, we found that histone H1 differentially enhances ligation reaction of DNA double-strand breaks (DSB). Histone H1 stimulated exclusively inter-molecular ligation reaction of DSB with DNA ligase IIIbeta and IV, whereas HMGB1 enhanced mainly intra-molecular ligation reaction. Electron microscopy of direct DNA-protein interaction without chemical cross-linking visualized that HMGB1 bends and loops linear DNA to form compact DNA structure and that histone H1 is capable of assembling DNA in tandem arrangement with occasional branches. These results suggest that differences in the enhancement of DNA ligation reaction are due to those in alteration of DNA configuration induced by these two linker proteins. HMGB1 and histone H1 may function in non-homologous end-joining of DSB repair and V(D)J recombination in different manners.
Topics: DNA; DNA Ligase ATP; DNA Ligases; HMGB1 Protein; Histones; Nucleosomes; Poly-ADP-Ribose Binding Proteins; Xenopus Proteins
PubMed: 11890703
DOI: 10.1006/bbrc.2002.6647 -
Journal of Molecular Biology Apr 2014High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize...
High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab.umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.
Topics: Base Sequence; DNA; DNA Ligases; Gene Library; Molecular Biology; Software; Thermodynamics
PubMed: 24518657
DOI: 10.1016/j.jmb.2014.02.003 -
Methods in Molecular Biology (Clifton,... 2014Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter...
Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.
Topics: Base Pair Mismatch; Base Sequence; Chromatography, High Pressure Liquid; DNA; Models, Molecular; Nucleic Acid Conformation; Peptide Nucleic Acids; Polymerase Chain Reaction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature
PubMed: 24297356
DOI: 10.1007/978-1-62703-553-8_11 -
Methods in Molecular Biology (Clifton,... 2017In situ ligation (ISL) is a simple and specific technique for apoptosis labeling in tissue sections. In its most economical version ISL uses ordinary PCR-labeled DNA...
In situ ligation (ISL) is a simple and specific technique for apoptosis labeling in tissue sections. In its most economical version ISL uses ordinary PCR-labeled DNA fragments as probes. In tissue sections these makeshift probes are ligated to apoptotic DNA breaks by T4 DNA ligase. The approach can selectively label 5'PO DNA breaks with blunt ends, and is the histological equivalent of electrophoretic apoptotic ladder detection. The main drawback of this technique is its low speed, as it requires 18 h-incubation for efficient labeling. Here, we describe an easy modification of ISL which reduces the incubation time to 1 h and converts ISL into a rapid detection method taking ~3 h overall. Signal enhancement is achieved by a new type of isothermal amplification reaction which generates "zebra tails"- long and labeled extensions of the probes attached to DNA breaks.
Topics: Apoptosis; DNA; DNA Breaks; Humans; In Situ Nick-End Labeling; Molecular Probe Techniques; Polymerase Chain Reaction; Tissue Fixation
PubMed: 28710763
DOI: 10.1007/978-1-4939-7187-9_15 -
Analytical Biochemistry Nov 2002We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp...
We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.
Topics: Base Composition; Base Sequence; DNA; DNA Ligases; DNA Mutational Analysis; DNA Primers; DNA, Neoplasm; Databases, Factual; Electrophoresis, Capillary; Gene Amplification; Genome, Human; Hot Temperature; Humans; Nucleic Acid Denaturation; Oligonucleotides; Osmolar Concentration; Point Mutation; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 12423636
DOI: 10.1016/s0003-2697(02)00383-4 -
Biochimica Et Biophysica Acta Nov 1983After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4,...
After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4',5'-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, lambda DNA to be ligated was as follows: lambda DNA = XP12 DNA greater than SP82 DNA approximately equal to nonglucosylated T4 DNA greater than T4 DNA = PBSI1 DNA much greater than SP15 DNA. Taq I-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and lambda DNAs was equally efficient. We conclude that the poor ligation of Taq I fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.
Topics: Bacteriophages; DNA Ligases; DNA, Viral; Polynucleotide Ligases; Structure-Activity Relationship; Substrate Specificity
PubMed: 6652091
DOI: 10.1016/0167-4781(83)90064-7