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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 -
The FEBS Journal Dec 2005T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli...
T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.
Topics: DNA Ligases; DNA, Circular; DNA, Single-Stranded; Oligonucleotides; Polymerase Chain Reaction
PubMed: 16302964
DOI: 10.1111/j.1742-4658.2005.04954.x -
The Journal of Nutritional Biochemistry Feb 2022Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging...
Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation.
Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging evidence suggests that gut microbiome and gut-derived metabolites play a key role in NP. We evaluated the effects of two ginger root extracts rich in gingerols (GEG) and shogaols (SEG) on pain sensitivity, anxiety-like behaviors, circulating cell-free mitochondrial DNA (ccf-mtDNA), gut microbiome composition, and fecal metabolites in rats with NP. Sixteen male rats were divided into four groups: sham, spinal nerve ligation (SNL), SNL+0.75%GEG in diet, and SNL+0.75%SEG in diet groups for 30 days. Compared to SNL group, both SNL+GEG and SNL+SEG groups showed a significant reduction in pain- and anxiety-like behaviors, and ccf-mtDNA level. Relative to the SNL group, both SNL+GEG and SNL+SEG groups increased the relative abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae, and Anaerovoracaceae, but decreased that of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio, Desulfovibrio, Anaerofilum, Eubacterium siraeum group, RF39, UCG-005, Lachnospiraceae NK4A136 group, Acetatifactor, Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae. GEG and SEG had differential effects on gut-derived metabolites. Compared to SNL group, SNL+GEG group had higher level of 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0∼1,10∼.0∼3,7∼]hexadec-9-en-5-one, while SNL+SEG group had higher level for (±)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate. In conclusion, ginger is a promising functional food in the management of NP, and further investigations are necessary to assess the role of ginger on gut-brain axis in pain management.
Topics: Animals; Bacteria; Catechols; DNA, Mitochondrial; Dietary Supplements; Fatty Alcohols; Feces; Gastrointestinal Microbiome; Gastrointestinal Tract; Zingiber officinale; Ligation; Male; Neuralgia; Pain Management; Plant Extracts; Rats; Rats, Sprague-Dawley; Spinal Nerves
PubMed: 34748918
DOI: 10.1016/j.jnutbio.2021.108904 -
BMC Molecular Biology Dec 2016Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of...
BACKGROUND
Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls.
RESULTS
To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40-1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41-2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76-1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145).
CONCLUSIONS
Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate multiple and complex normalization controls and provide accurate calculation of proximity-based fragment ligation frequency. Therefore, we recommend 3C-dPCR as a preferred method for sensitive detection of low frequency chromatin interactions.
Topics: Cell Line, Tumor; Chromatin; DNA; Genes, myc; Genetic Loci; Humans; Male; Microfilament Proteins; Nuclear Proteins; Polymerase Chain Reaction; Prostatic Neoplasms
PubMed: 27923366
DOI: 10.1186/s12867-016-0076-6 -
Nucleic Acids Research Nov 1998Several different computational problems have been solved using DNA as a medium. However, the DNA computations that have so far been carried out have examined a...
Several different computational problems have been solved using DNA as a medium. However, the DNA computations that have so far been carried out have examined a relatively small number of possible sequence solutions in order to find correct sequence solutions. We have encoded a search algorithm in DNA that required the evaluation of >16 000 000 possible sequence solutions in order to find a single, correct sequence solution. Experimental evaluation of the search algorithm revealed bounds for the accuracies of answers to other large, computationally complex problems and suggested methods for the optimization of DNA computations in general. Short oligonucleotide substrates performed substantially better than longer substrates. Large, computationally complex problems whose evaluation requires hybridization and ligation can likely best be encoded and evaluated using short oligonucleotides at mesophilic temperatures.
Topics: Algorithms; Base Sequence; DNA; DNA Primers; Models, Genetic; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides
PubMed: 9801320
DOI: 10.1093/nar/26.22.5203 -
Nucleic Acids Research 2007The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities....
The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Ligase ATP; DNA Ligases; DNA Repair Enzymes; DNA, Single-Stranded; DNA-Binding Proteins; Humans; Magnesium
PubMed: 17717001
DOI: 10.1093/nar/gkm579 -
Biopolymers Jan 2021We present cognate base pair selectivity in template-dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds-Pa. T4 DNA ligase efficiently...
We present cognate base pair selectivity in template-dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds-Pa. T4 DNA ligase efficiently recognizes the Ds-Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base-pair selectivity, relative to the hydrogen-bond interactions between pairing bases. The efficient ligation using Ds-Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi-synthetic organisms containing UBPs.
Topics: Base Pairing; DNA; DNA Ligases; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Nucleotides
PubMed: 33156531
DOI: 10.1002/bip.23407 -
Nature Methods Mar 2012High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform...
High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.
Topics: Base Sequence; DNA; DNA Ligases; GC Rich Sequence; High-Throughput Nucleotide Sequencing; Magnetics; Nucleic Acid Conformation; Nucleic Acid Hybridization; Templates, Genetic
PubMed: 22406857
DOI: 10.1038/nmeth.1925 -
The Journal of Biological Chemistry Nov 2004The repair of DNA double-strand breaks is critical for maintaining genetic stability. In the non-homologous end-joining pathway, DNA ends are brought together by...
The repair of DNA double-strand breaks is critical for maintaining genetic stability. In the non-homologous end-joining pathway, DNA ends are brought together by end-bridging factors. However, most in vivo DNA double-strand breaks have terminal structures that cannot be directly ligated. Thus, the DNA ends are aligned using short regions of sequence microhomology followed by processing of the aligned DNA ends by DNA polymerases and nucleases to generate ligatable termini. Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structure-specific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1. In this study, we demonstrated that FEN-1(Rad27) physically and functionally interacted with both Pol4 and Dnl4/Lif1 and that together these proteins coordinately processed and joined DNA molecules with incompatible 5' ends. Because Pol4 also interacts with Dnl4/Lif1, our results have revealed a series of pair-wise interactions among the factors that complete the repair of DNA double-strand breaks by non-homologous end-joining and provide a conceptual framework for delineating the end-processing reactions in higher eukaryotes.
Topics: Acetyltransferases; Base Sequence; DNA; DNA Damage; DNA Ligase ATP; DNA Ligases; DNA Polymerase beta; DNA Repair; DNA-Directed DNA Polymerase; Membrane Proteins; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Nucleic Acid
PubMed: 15342630
DOI: 10.1074/jbc.M404492200 -
Archives of Pathology & Laboratory... Dec 1999To review the advances in clinically useful molecular biological techniques and to identify their applications in clinical practice, as presented at the Eighth Annual... (Review)
Review
OBJECTIVES
To review the advances in clinically useful molecular biological techniques and to identify their applications in clinical practice, as presented at the Eighth Annual William Beaumont Hospital Symposium.
DATA SOURCES
The 10 manuscripts submitted were reviewed, and their major findings were compared with literature on the same topic.
STUDY SELECTION
Two manuscripts addressed specimen (nucleic acid) stability, 2 described novel analytic approaches, 3 discussed detection of B- or T-cell clonality in lymphoproliferative disorders, and 3 reported the frequency of a variety of genetic polymorphisms found in cardiac disorders.
DATA SYNTHESIS
DNA from dried blood spots is stable and may be purified rapidly for amplification and mutation analysis. RNA is much less stable, and a variety of methods may be used to reduce ribonuclease degradation of enteroviral RNA. False-negative reactions may be reduced by genomic amplification of ligated padlock probes by cascade rolling circle or polymerase chain reaction. A multiplex polymerase chain method using fluorescence-labeled products that separate both the wild-type and mutant hemochromatosis gene alleles by capillary gel electrophoresis represents another approach for detecting the 2 major missense mutations (C282Y and H63D) in hemochromatosis. Southern blotting and polymerase chain reaction have been used to detect B- and T-cell clonality in lymphoproliferative diseases, including mantle cell lymphoma and lymphoma of the breast. Genetic polymorphisms in a variety of coagulation factors and platelet glycoprotein IIIa are associated with ischemic heart disease.
CONCLUSIONS
As the Human Genome Project continues to define disease-associated mutations, the number of clinically useful molecular pathologic techniques and assays will expand. Clinical outcome analysis is still required to document a decrease in the patient's length of stay to offset the cost of introducing molecular biological assays in the routine clinical pathology laboratory.
Topics: Clinical Laboratory Techniques; DNA; Genetic Techniques; Humans
PubMed: 10583918
DOI: 10.5858/1999-123-1151-DTITCL