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Nucleic Acids Research Mar 1990DNA methylation at specific sites is most frequently studied by use of methylation-sensitive restriction endonucleases and Southern blotting. We report here that the...
DNA methylation at specific sites is most frequently studied by use of methylation-sensitive restriction endonucleases and Southern blotting. We report here that the sensitivity of this method can be increased several-hundred-fold by applying a ligation-mediated polymerase chain reaction (LM-PCR) procedure following enzyme treatment. DNA is cleaved simultaneously with two restriction enzymes, one sensitive and one insensitive to methylation. After cleavage, a gene-specific oligonucleotide primer is used for primer extension, followed by linker ligation and then conventional PCR. Using this technique, we demonstrate that DNA from 100 cells (about 0.6 ng) can be prepared and qualitatively analyzed for methylation at sites in an X-linked CpG island, and 50 ng of DNA can be analyzed quantitatively. A site 23 bp downstream of the major transcription start site of human phosphoglycerate kinase-1 (PGK-1) is 52 +/- 7 percent methylated in DNA from female blood and greater than 98 percent unmethylated in DNA from male blood or HeLa cells. This method detects quantitatively specific breaks in either double stranded or single stranded DNA. Thus new assays for enzymes and DNA structure can be devised.
Topics: Animals; Base Sequence; Cell Nucleus; Cricetinae; Cricetulus; DNA; DNA Damage; DNA Restriction Enzymes; Humans; Hybrid Cells; Methylation; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Oligonucleotide Probes; Polymerase Chain Reaction; Restriction Mapping; X Chromosome
PubMed: 2158077
DOI: 10.1093/nar/18.6.1435 -
Biochimica Et Biophysica Acta Dec 2011Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in...
Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.
Topics: Blotting, Southern; Blotting, Western; Bone Neoplasms; DNA; DNA Ligase ATP; DNA Ligases; DNA Replication; DNA, Mitochondrial; Flap Endonucleases; Humans; Osteosarcoma; Poly-ADP-Ribose Binding Proteins; Ribonuclease H; Thymidine Kinase; Tumor Cells, Cultured; Xenopus Proteins
PubMed: 21878356
DOI: 10.1016/j.bbamcr.2011.08.008 -
Nucleic Acids Research Jan 2017RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to...
RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.
Topics: Adenosine Diphosphate; Coenzymes; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA, Fungal; DNA, Single-Stranded; Plasmids; Rad51 Recombinase; Rec A Recombinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 27794044
DOI: 10.1093/nar/gkw998 -
PLoS Computational Biology Oct 2021Hi-C is a sample preparation method that enables high-throughput sequencing to capture genome-wide spatial interactions between DNA molecules. The technique has been...
Hi-C is a sample preparation method that enables high-throughput sequencing to capture genome-wide spatial interactions between DNA molecules. The technique has been successfully applied to solve challenging problems such as 3D structural analysis of chromatin, scaffolding of large genome assemblies and more recently the accurate resolution of metagenome-assembled genomes (MAGs). Despite continued refinements, however, preparing a Hi-C library remains a complex laboratory protocol. To avoid costly failures and maximise the odds of successful outcomes, diligent quality management is recommended. Current wet-lab methods provide only a crude assay of Hi-C library quality, while key post-sequencing quality indicators used have-thus far-relied upon reference-based read-mapping. When a reference is accessible, this reliance introduces a concern for quality, where an incomplete or inexact reference skews the resulting quality indicators. We propose a new, reference-free approach that infers the total fraction of read-pairs that are a product of proximity ligation. This quantification of Hi-C library quality requires only a modest amount of sequencing data and is independent of other application-specific criteria. The algorithm builds upon the observation that proximity ligation events are likely to create k-mers that would not naturally occur in the sample. Our software tool (qc3C) is to our knowledge the first to implement a reference-free Hi-C QC tool, and also provides reference-based QC, enabling Hi-C to be more easily applied to non-model organisms and environmental samples. We characterise the accuracy of the new algorithm on simulated and real datasets and compare it to reference-based methods.
Topics: Algorithms; Animals; Chromosome Mapping; DNA; Gene Library; Genomics; High-Throughput Nucleotide Sequencing; Humans; Quality Control; Software; Turtles
PubMed: 34634030
DOI: 10.1371/journal.pcbi.1008839 -
PloS One 2013The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA... (Comparative Study)
Comparative Study
The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.
Topics: Cloning, Molecular; DNA; Genes, Reporter; Genetic Vectors; Plasmids
PubMed: 24376768
DOI: 10.1371/journal.pone.0083888 -
Methods in Molecular Biology (Clifton,... 2012The formation and repair of DNA damage at specific locations in the genome is modulated by DNA sequence context, by DNA cytosine-5 methylation patterns, by the...
The formation and repair of DNA damage at specific locations in the genome is modulated by DNA sequence context, by DNA cytosine-5 methylation patterns, by the transcriptional status of the locus and by proteins associated with the DNA. The only method currently available to allow precise sequence mapping of DNA lesions in mammalian cells is the ligation-mediated polymerase chain reaction (LM-PCR) technique. We provide an update on technical details of LM-PCR. LM-PCR can be used, for example, for mapping of ultraviolet (UV) light-induced DNA photoproducts such as cyclobutane pyrimidine dimers.
Topics: Animals; Base Sequence; Biotinylation; DNA; DNA Cleavage; DNA Damage; DNA Primers; DNA Repair; Deoxyribonuclease (Pyrimidine Dimer); Magnets; Mice; Microspheres; Polymerase Chain Reaction; Pyrimidine Dimers; Streptavidin; Ultraviolet Rays
PubMed: 22941605
DOI: 10.1007/978-1-61779-998-3_14 -
PloS One 2016DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA...
DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.
Topics: Catalysis; DNA; DNA Ligases; DNA Replication; In Vitro Techniques; Kinetics
PubMed: 26954034
DOI: 10.1371/journal.pone.0150802 -
Journal of Biochemistry Jul 2002Scanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library...
Scanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library containing all possible single-residue mutants of the protein of interest. The practical application of this approach is currently limited by the large number and cost of the required 30-35mer oligonucleotides. As an alternative, we studied the ligation of shorter DNA oligonucleotides (6-11mer) containing a degenerate binding site and a desired mutation mismatch to a nested set of megaprimers annealed to the gene of interest. T4 DNA ligase was able to perform this task, and the obtained ligation products were elongated by DNA polymerase. The effectiveness of ligation depends on the length of the random binding site of the mutagenic oligonucleotide, on its molar excess over the template-primer complex and on the position of the mismatching tri-nucleotide insert with respect to the joining site. The secondary structure of the DNA template close to the joining site also influences the ligation yield. Mismatching oligonucleotides, protected by a 3'-phosphate group, were joined to a nested set of megaprimers, the latter being obtained by a novel procedure called reversible chain termination, i.e., termination of the dsDNA synthesis with ddNTP followed by the subsequent removal of the incorporated ddNMP with exonuclease III. T7 sequenase 2.0 DNA polymerase elongated the ligation products after the 3'-phosphate protection group was removed with T4 polynucleotide kinase, resulting in the incorporation of a specific tri-nucleotide mismatch into dsDNA. This sequence of reactions serves as the basis for a novel scanning mutagenesis procedure.
Topics: Bacteriophage T4; Base Pair Mismatch; Binding Sites; DNA; DNA Ligases; DNA Primers; DNA-Directed DNA Polymerase; Exodeoxyribonucleases; Kinetics; Mutagenesis; Oligodeoxyribonucleotides; Polynucleotide 5'-Hydroxyl-Kinase; Substrate Specificity
PubMed: 12097171
DOI: 10.1093/oxfordjournals.jbchem.a003192 -
ELife Nov 2018Recombination, the exchange of information between different genetic polymer strands, is of fundamental importance in biology for genome maintenance and genetic...
Recombination, the exchange of information between different genetic polymer strands, is of fundamental importance in biology for genome maintenance and genetic diversification and is mediated by dedicated recombinase enzymes. Here, we describe an innate capacity for non-enzymatic recombination (and ligation) in random-sequence genetic oligomer pools. Specifically, we examine random and semi-random eicosamer (N) pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids). While DNA, ANA and HNA pools proved inert, RNA (and to a lesser extent AtNA) pools displayed diverse modes of spontaneous intermolecular recombination, connecting recombination mechanistically to the vicinal ring cis-diol configuration shared by RNA and AtNA. Thus, the chemical constitution that renders both susceptible to hydrolysis emerges as the fundamental determinant of an innate capacity for recombination, which is shown to promote a concomitant increase in compositional, informational and structural pool complexity and hence evolutionary potential.
Topics: Base Pairing; Base Sequence; DNA; Kinetics; Models, Molecular; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Oligoribonucleotides; Polysaccharides; RNA; Recombination, Genetic; Solutions; Sugar Alcohols; Thermodynamics
PubMed: 30461419
DOI: 10.7554/eLife.43022 -
Molecular and Cellular Biology Jan 1994Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made...
Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.
Topics: Adenoviridae; Animals; Base Sequence; Cell Line; DNA; DNA Damage; DNA Ligases; DNA Repair; DNA, Viral; Humans; Microscopy, Electron; Models, Genetic; Proteins; Recombination, Genetic; Transfection; Tumor Cells, Cultured
PubMed: 8264583
DOI: 10.1128/mcb.14.1.156-169.1994