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Nature Chemistry Apr 2018The evolution of sequence-defined synthetic polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has the potential to...
The evolution of sequence-defined synthetic polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has the potential to generate new classes of receptors, catalysts and materials. Here we describe a ligase-mediated DNA-templated polymerization and in vitro selection system to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks that contain eight chemically diverse side chains on a DNA backbone. Through iterated cycles of polymer translation, selection and reverse translation, we discovered HFNAPs that bind proprotein convertase subtilisin/kexin type 9 (PCSK9) and interleukin-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (K = 3 nM). This evolved polymer potently inhibited the binding between PCSK9 and the low-density lipoprotein receptor. Structure-activity relationship studies revealed that specific side chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires.
Topics: Base Sequence; DNA; DNA Ligases; Humans; Polymers
PubMed: 29507367
DOI: 10.1038/s41557-018-0008-9 -
PloS One 2020DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly...
DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. For example, selection of the DNA sequences at fusion sites between fragments is based on broad assembly guidelines or pre-vetted sets of junctions, rather than being customized for a particular application or cloning project. To facilitate the design of robust assembly reactions, we developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most commonly used Type IIS restriction enzymes that generate three-base and four-base overhangs. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. MoClo) as well as the formation of robust new high-fidelity standards.
Topics: DNA; DNA Ligases; DNA Restriction Enzymes; Nucleotides; Synthetic Biology
PubMed: 32877448
DOI: 10.1371/journal.pone.0238592 -
Journal of Biochemistry Jan 1987High concentrations of the nonspecific macromolecule polyethylene glycol 6000 (PEG 6000) enabled DNA ligases I and II from calf thymus to catalyze intermolecular...
High concentrations of the nonspecific macromolecule polyethylene glycol 6000 (PEG 6000) enabled DNA ligases I and II from calf thymus to catalyze intermolecular blunt-end ligation of duplex DNA. Intermolecular cohesive-end ligation with these enzymes was markedly stimulated in the presence of 10-16% (w/v) PEG 6000. The effect of PEG 6000 (4-16%) on the sealing of single-stranded breaks in duplex DNA with DNA ligases I and II was not appreciably stimulatory but rather inhibitory. PEG 6000 (15%) enhanced more twofold the rate of DNA ligase II-AMP complex formation, but moderately suppressed the rate of formation of DNA ligase 1-AMP complex. Polyamines and KCl inhibited blunt-end and cohesive-end ligations with DNA ligases I and II in the presence of PEG 6000.
Topics: Animals; Cattle; DNA Ligase ATP; DNA Ligases; Polyethylene Glycols; Polynucleotide Ligases; Potassium Chloride; Spermine; Thymus Gland
PubMed: 3571204
DOI: 10.1093/oxfordjournals.jbchem.a121895 -
The Journal of Biological Chemistry Aug 1998The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch...
The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites for self-association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol beta interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol beta did not. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain.
Topics: Chromatography, Affinity; DNA Ligase ATP; DNA Ligases; DNA Polymerase beta; DNA Repair; Humans; Protein Binding; Protein Conformation; Recombinant Proteins; Static Electricity; Thermodynamics; Ultracentrifugation
PubMed: 9685411
DOI: 10.1074/jbc.273.32.20540 -
Nucleic Acids Research Sep 2018DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5'P and 3'OH ends in an adenylation-dependent manner....
DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5'P and 3'OH ends in an adenylation-dependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker.
Topics: Alteromonas; Amino Acid Substitution; Bacterial Proteins; Binding Sites; Catalytic Domain; Conserved Sequence; Crystallography, X-Ray; DNA Breaks, Single-Stranded; DNA Ligases; DNA, Bacterial; Genes, Synthetic; Models, Molecular; Mutagenesis, Site-Directed; Point Mutation; Protein Binding; Protein Conformation; Protein Domains; Protein Stability; Sequence Alignment; Structure-Activity Relationship; Substrate Specificity
PubMed: 30007325
DOI: 10.1093/nar/gky622 -
Nucleic Acids Research Jul 2020Allostery is comprehensively studied for natural macromolecules, such as proteins and nucleic acids. Here, we present controllable allostery of synthetic DNA...
Allostery is comprehensively studied for natural macromolecules, such as proteins and nucleic acids. Here, we present controllable allostery of synthetic DNA nanostructure-enzyme systems. Rational designs of the synthetic allosteric systems are based on an in-depth understanding of allosteric sites with several types of strand placements, whose varying stacking strengths determine the local conformation and ultimately lead to a gradient level of allosteric transition. When enzymes in a molecular cloning toolbox such as DNA polymerase, exonuclease and ligase are applied to treat the allosteric sites, the resulting local conformational changes propagate through the entire structure for a global allosteric transition.
Topics: Allosteric Regulation; DNA; DNA Ligases; DNA-Directed DNA Polymerase; Exodeoxyribonucleases; Nanostructures; Nucleic Acid Conformation
PubMed: 32526030
DOI: 10.1093/nar/gkaa488 -
Nucleic Acids Research Oct 1998A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with...
A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with 5'-hydroxyl termini or 3'-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, i.e. 5'-phosphate and 3'-hydroxyl termini. To further assess this possibility, we compared the activity of the 3'-phosphatase of purified calf thymus polynucleotide kinase towards a variety of substrates. The rate of removal of 3'-phosphate groups from nicked or short (1 nt) gapped sites in double-stranded DNA was observed to be similar to that of 3'-phosphate groups from single-stranded substrates. Thus this activity of polynucleotide kinase does not appear to be influenced by steric accessibility of the phosphate group. We subsequently demonstrated that the concerted reactions of polynucleotide kinase and purified human DNA ligase I could efficiently repair DNA nicks possessing 3'-phosphate and 5'-hydroxyl termini, and similarly the combination of these two enzymes together with purified rat DNA polymerase beta could seal a strand break with a 1 nt gap. With a substrate containing a nick bounded by 3'- and 5'-OH termini, the rate of gap filling by polymerase beta was significantly enhanced in the presence of polynucleotide kinase and ATP, indicating the positive influence of 5'-phosphorylation. The reaction was further enhanced by addition of DNA ligase I to the reaction mixture. This is due, at least in part, to an enhancement by DNA ligase I of the rate of 5'-phosphorylation catalyzed by polynucleotide kinase.
Topics: Animals; Base Sequence; DNA Ligases; DNA Repair; DNA-Directed DNA Polymerase; Humans; Molecular Sequence Data; Phosphorylation; Polynucleotide 5'-Hydroxyl-Kinase; Rats
PubMed: 9742240
DOI: 10.1093/nar/26.19.4395 -
Nature Communications Nov 2023Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In...
Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In this study, bdelloid rotifer Adineta vaga is used as a model to decipher the molecular basis of their extreme tolerance. Proteomic analysis shows that a specific DNA ligase, different from those usually involved in DNA repair in eukaryotes, is strongly over-represented upon ionizing radiation. A phylogenetic analysis reveals its orthology to prokaryotic DNA ligase E, and its horizontal acquisition by bdelloid rotifers and plausibly other eukaryotes. The fungus Mortierella verticillata, having a single copy of this DNA Ligase E homolog, also exhibits an increased radiation tolerance with an over-expression of this DNA ligase E following X-ray exposure. We also provide evidence that A. vaga ligase E is a major contributor of DNA breaks ligation activity, which is a common step of all important DNA repair pathways. Consistently, its heterologous expression in human cell lines significantly improves their radio-tolerance. Overall, this study highlights the potential of horizontal gene transfers in eukaryotes, and their contribution to the adaptation to extreme conditions.
Topics: Animals; Humans; Eukaryota; Phylogeny; DNA Ligases; Ligases; Proteomics; Rotifera; DNA Damage; DNA Ligase ATP
PubMed: 37993452
DOI: 10.1038/s41467-023-43075-8 -
Analytical Chemistry Oct 2008This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in...
This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.
Topics: DNA Breaks, Single-Stranded; DNA Ligases; DNA, Superhelical; Electrochemical Techniques; Escherichia coli
PubMed: 18778033
DOI: 10.1021/ac801268p -
Nucleic Acids Research Jul 2016We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method...
We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.
Topics: Animals; DNA Ligases; DNA Probes; Humans; Mice; MicroRNAs; Nucleic Acid Hybridization; Rats; Real-Time Polymerase Chain Reaction; Viral Proteins
PubMed: 27154271
DOI: 10.1093/nar/gkw399