-
Accounts of Chemical Research Aug 2012Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1... (Review)
Review
Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have potential therapeutic applications. They have extremely high thermodynamic stability, have increased resistance to enzymatic degradation, and have been investigated as decoys for regulatory proteins. For potential nanotechnology applications, we have synthesized double stranded DNA catenanes by click ligation. Other researchers have studied covalently fixed multistranded DNA constructs including triplexes and quadruplexes.
Topics: Biology; Click Chemistry; DNA; Humans; Nanotechnology; RNA; Transcription, Genetic
PubMed: 22439702
DOI: 10.1021/ar200321n -
Nucleic Acids Research May 2022DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To...
DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.
Topics: DNA; DNA Ligases; Humans; Purines
PubMed: 35438779
DOI: 10.1093/nar/gkac241 -
Scientific Reports Mar 2018Most members of the poly(ADP-ribose)polymerase family, PARP family, have a catalytic activity that involves the transfer of ADP-ribose from a beta-NAD+-molecule to...
Most members of the poly(ADP-ribose)polymerase family, PARP family, have a catalytic activity that involves the transfer of ADP-ribose from a beta-NAD+-molecule to protein acceptors. It was recently discovered by Talhaoui et al. that DNA-dependent PARP1 and PARP2 can also modify DNA. Here, we demonstrate that DNA-dependent PARP3 can modify DNA and form a specific primed structure for further use by the repair proteins. We demonstrated that gapped DNA that was ADP-ribosylated by PARP3 could be ligated to double-stranded DNA by DNA ligases. Moreover, this ADP-ribosylated DNA could serve as a primed DNA substrate for PAR chain elongation by the purified proteins PARP1 and PARP2 as well as by cell-free extracts. We suggest that this ADP-ribose modification can be involved in cellular pathways that are important for cell survival in the process of double-strand break formation.
Topics: Cell Cycle Proteins; Cell-Free System; DNA; DNA Breaks, Double-Stranded; Humans; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases
PubMed: 29520010
DOI: 10.1038/s41598-018-22673-3 -
Nucleic Acids Research Feb 2014Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of...
Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.
Topics: DNA; DNA Ligases; Kinetics; RNA; Viral Proteins
PubMed: 24203707
DOI: 10.1093/nar/gkt1032 -
RNA Biology 2014Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose...
Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.
Topics: Animals; DNA; DNA Damage; DNA Repair; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA-Binding Proteins; Humans; Models, Genetic; Models, Molecular; Molecular Structure; Nuclear Proteins; Nucleic Acid Conformation; Protein Binding; Protein Structure, Tertiary; RNA; Ribonucleotides
PubMed: 25692233
DOI: 10.4161/15476286.2014.992283 -
Acta Biochimica Polonica 2016A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods...
A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal.
Topics: Amplified Fragment Length Polymorphism Analysis; DNA; Microbiota; Polymerase Chain Reaction
PubMed: 26885774
DOI: 10.18388/abp.2015_1192 -
Nucleic Acids Research Feb 2009Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix.... (Review)
Review
Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA cleavage mediated by the enzyme.
Topics: Antineoplastic Agents; DNA; DNA Cleavage; DNA Damage; DNA Topoisomerases, Type II; Diet; Humans; Leukemia; Quinones; Topoisomerase II Inhibitors
PubMed: 19042970
DOI: 10.1093/nar/gkn937 -
Nature Communications Feb 2024Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that...
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.
Topics: DNA Ligase ATP; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA Ligases; DNA
PubMed: 38341432
DOI: 10.1038/s41467-024-45553-z -
Nucleic Acids Research May 2020Class-II AP-endonuclease (XthA) and NAD+-dependent DNA ligase (LigA) are involved in initial and terminal stages of bacterial DNA base excision repair (BER),...
M. tuberculosis class II apurinic/ apyrimidinic-endonuclease/3'-5' exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair.
Class-II AP-endonuclease (XthA) and NAD+-dependent DNA ligase (LigA) are involved in initial and terminal stages of bacterial DNA base excision repair (BER), respectively. XthA acts on abasic sites of damaged DNA to create nicks with 3'OH and 5'-deoxyribose phosphate (5'-dRP) moieties. Co-immunoprecipitation using mycobacterial cell-lysate, identified MtbLigA-MtbXthA complex formation. Pull-down experiments using purified wild-type, and domain-deleted MtbLigA mutants show that LigA-XthA interactions are mediated by the BRCT-domain of LigA. Small-Angle-X-ray scattering, 15N/1H-HSQC chemical shift perturbation experiments and mutational analysis identified the BRCT-domain region that interacts with a novel 104DGQPSWSGKP113 motif on XthA for complex-formation. Isothermal-titration calorimetry experiments show that a synthetic peptide with this sequence interacts with MtbLigA and disrupts XthA-LigA interactions. In vitro assays involving DNA substrate and product analogs show that LigA can efficiently reseal 3'OH and 5'dRP DNA termini created by XthA at abasic sites. Assays and SAXS experiments performed in the presence and absence of DNA, show that XthA inhibits LigA by specifically engaging with the latter's BRCT-domain to prevent it from encircling substrate DNA. Overall, the study suggests a coordinating function for XthA whereby it engages initially with LigA to prevent the undesirable consequences of futile cleavage and ligation cycles that might derail bacterial BER.
Topics: DNA; DNA Cleavage; DNA Ligases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Mycobacterium tuberculosis; Protein Conformation; Protein Interaction Domains and Motifs
PubMed: 32232338
DOI: 10.1093/nar/gkaa188 -
Biophysical Journal Nov 2022DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied...
DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.
Topics: DNA; Nanostructures; Microscopy, Atomic Force; Molecular Dynamics Simulation; Elasticity; Nucleic Acid Conformation
PubMed: 36181269
DOI: 10.1016/j.bpj.2022.09.036