Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Selective Ligase-Based Sample Processing-Free Discrimination and Detection of Site-Specific DNA 5-Hydroxymethylcytosine.

Accurate detection of site-specific 5-hydroxymethylcytosine (5hmC) in genomic DNA is of great significance, but it is technically challenging to directly distinguish very low levels of 5hmC from their abundant cytosine/5-methylcytosine (C/5mC) analogues. Herein, we wish to propose a selective ligase-mediated mechanism (SLim) that can directly discriminate 5hmC from C/5mC with a high specificity without the use of any sample processing protocol. In this new design, we discovered that HiFi Taq DNA Ligase can well tolerate the mismatched 5hmC/A base-pairing and then effectively ligate the associated nicking site while the mismatched 5mC/A or C/A pairs cannot be recognized by HiFi Taq DNA Ligase, providing a new way for direct and selective discriminating 5hmC from its similar analogues. Ultrasensitive and selective quantification of site-specific 5hmC is realized by coupling the SLim with polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).

Accurate identification of 8-oxoguanine in RNA with single-nucleotide resolution using ligase-dependent qPCR.

8-oxoguanine (o8G), a prevalent oxidative modification in RNA induced by reactive oxygen species (ROS), plays a pivotal role in regulating RNA functions. Accurate detection and quantification of o8G modifications is critical to understanding their biological significance and potential as disease biomarkers, but effective detection methods remain limited. Here, we have developed a highly specific T3 DNA ligase-dependent qPCR assay that exploits the enzyme's ability to discriminate o8G from guanine (G) with single-nucleotide resolution. This method can detect o8G in RNA at levels as low as 500 fM, with an up to 18-fold higher selectivity for discriminating o8G from G. By simulating oxidative stress conditions in SH-SY5Y and HS683 cell lines treated with rotenone, we successfully identified site-specific o8G modifications in key miRNAs associated with neuroprotective responses, including miR-124, let-7a and miR-29a. The developed assay holds significant promise for the practical identification of o8G, facilitating its potential for detailed studies of o8G dynamics in various biological contexts and diseases.

Establishment of a nonradioactive DNA ligation assay and its applications in murine tissues.

As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.

Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.

Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.

Four Parallel Pathways in T4 Ligase-Catalyzed Repair of Nicked DNA with Diverse Bending Angles.

The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.

1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA.

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 ß-sheets, closely resembling the typical structural topology of most BRCT domains.

R2D ligase: Unveiling a novel DNA ligase with surprising DNA-to-RNA ligation activity.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO4 2- end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.

Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining.

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.

Unfilled gaps by polß lead to aberrant ligation by LIG1 at the downstream steps of base excision repair pathway.

Base excision repair (BER) involves the tightly coordinated function of DNA polymerase ß (polß) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polß to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polß leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polß to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polß and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.

Formation of an instantaneous nick for highly efficient adenylation of oligonucleotides by ligase without subsequent jointing.

By forming a nick at the adenylation site instantaneously, nucleic acids are efficiently adenylated by T4 DNA ligase. The subsequent ligation is successfully suppressed in terms of rapid conversion of the instantaneous nick to a more stable gap. It is helpful to understand enzymatic ligation dynamics, and the adenylated products can be used for various practical applications.

Endonuclease IV and T4 ligase enhanced detection of mutations in low abundance.

We propose a mutant detection approach based on endonuclease IV and DNA ligase in combination with qPCR. The enzymes functioned cooperatively to facilitate PCR for low abundance DNA detection. We demonstrate that our approach can distinguish mutations as low as 0.01%, indicating the potential application of this strategy in early cancer diagnosis.

A role for the ATP-dependent DNA ligase lig E of Neisseria gonorrhoeae in biofilm formation.

BACKGROUND: The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype. RESULTS: This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation. CONCLUSIONS: These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.

Mammalian DNA ligases; roles in maintaining genome integrity.

The joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication, cytosine demethylation during differentiation, gene rearrangement in the immune system and germ cell development. In addition, they are generated either directly by a DNA damaging agent or indirectly due to damage excision during repair. Breaks are joined by a DNA ligase that catalyzes phosphodiester bond formation at DNA nicks with 3' hydroxyl and 5' phosphate termini. Three human genes encode ATP-dependent DNA ligases. These enzymes have a conserved catalytic core consisting of three subdomains that encircle nicked duplex DNA during ligation. The DNA ligases are targeted to different nuclear DNA transactions by specific protein-protein interactions. Both DNA ligase IIIα and DNA ligase IV form stable complexes with DNA repair proteins, XRCC1 and XRCC4, respectively. There is functional redundancy between DNA ligase I and DNA ligase IIIα in DNA replication, excision repair and single-strand break repair. Although DNA ligase IV is a core component of the major double-strand break repair pathway, non-homologous end joining, the other enzymes participate in minor, alternative double-strand break repair pathways. In contrast to the nucleus, only DNA ligase IIIα is present in mitochondria and is essential for maintaining the mitochondrial genome. Human immunodeficiency syndromes caused by mutations in either LIG1 or LIG4 have been described. Preclinical studies with DNA ligase inhibitors have identified potentially targetable abnormalities in cancer cells and evidence that DNA ligases are potential targets for cancer therapy.

Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair.

Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase ß-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.

Horizontal acquisition of a DNA ligase improves DNA damage tolerance in eukaryotes.

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.

NAD+ mediated photoelectrochemical biosensor for histone deacetylase Sirt1 detection based on CuO-BiVO4-AgNCs heterojunction and hybridization chain reaction amplification.

BACKGROUND: Histone deacetylate Sirt1 has been involved in many important biological processes and is closely related to the occurrence and development of many diseases. Therefore, the accurate detection of Sirt1 is of great significance for the diagnosis and treatment of diseases caused by Sirt1 and the development of related drugs. RESULTS: In this work, a photoelectrochemical biosensor was developed for Sirt1 detection based on the NAD + mediated Sirt1 recognition and E. Coli DNA ligase activity. CuO-BiVO4p-n heterojunction was employed as the photoactive material, rolling circle amplification (RCA), hybridization chain reaction (HCR) and AgNCs were used as triple signal amplifications. As a bifunctional cofactor, NAD+ played a crucial role for Sirt1 detection, where the peptide deacetylation catalyzed by Sirt1 consumed NAD+, and the decreased amount of NAD + inhibited the activity of E. Coli DNA ligase, leading to the failure on RCA reaction, and improving the HCR reaction. Finally, AgNCs were generated using C-rich DNA as carrier. The surface plasmon effect of AgNCs and its heterojunction with CuO and BiVO4 accelerated the transfer rate of photogenerated carriers and improved the photocurrent signal. When the detection range was 0.001-200 nM, the detection limit of the biosensor was 0.76 pM (S/N = 3). SIGNIFICANCE: The applicability of the method was evaluated by studying the effects of known inhibitors nicotinamide and environmental pollutant halogenated carbazole on Sirt1 enzyme activity. The results showed that this method can be used as a new platform for screening Sirt1 enzyme inhibitors, and also provided a new biomarker for evaluating the ecotoxicological effects of environmental pollutants.

Homogeneous detection of viral nucleic acid via selective recognition proximity ligation and signal amplification with T7 transcription and CRISPR/Cas12a system.

The synthetic biology has employed the synthetic gene networks through engineering to construct various functions in biological systems. However, the use of gene circuits to create sensors for detecting low-abundance targets has been limited due to the lack of signal amplification strategies beyond direct output of detection signals. To address this issue, we introduce a novel method utilizing Selective Recognition Proximity Ligation and signal amplification with T7 Transcription and CRISPR/Cas12a system (SRPL-TraCs), which permits the incorporation of cell-free gene circuits with signal amplification and enables the construction of high-order cascade signal amplification strategy to detect biomarkers in homogeneous systems. Specifically, the SRPL-TraCs utilizes selective recognition proximity ligation with high-fidelity T4 DNA ligase and generates a unique crRNA via T7 transcription, along with target-activated Cas12a/crRNA system to achieve excellent specificity for HIV-1 DNA. With this straightforward synthetic biology-based method, the proposed SRPL-TraCs has the potential to detect numerous other interesting targets beyond the nucleic acids.

Codon usage bias analysis of the gene encoding NAD+-dependent DNA ligase protein of Invertebrate iridescent virus 6.

The genome of Invertebrate iridescent virus 6 (IIV6) contains a sequence that shows similarity to eubacterial NAD+-dependent DNA ligases. The 615-amino acid open reading frame (ORF 205R) consists of several domains, including an N-terminal domain Ia, followed by an adenylation domain, an OB-fold domain, a helix-hairpin-helix (HhH) domain, and a BRCT domain. Notably, the zinc finger domain, typically present in NAD+-dependent DNA ligases, is absent in ORF 205R. Since the protein encoded by ORF 205R (IIV6 DNA ligase gene) is involved in critical functions such as DNA replication, modification, and repair, it is crucial to comprehend the codon usage associated with this gene. In this paper, the codon usage bias (CUB) in DNA ligase gene of IIV6 and 11 reference iridoviruses was analyzed by comparing the nucleotide contents, relative synonymous codon usage (RSCU), effective number of codons (ENC), codon adaptation index (CAI), relative abundance of dinucleotides and other indices. Both the base content and the RCSU analysis indicated that the A- and T-ending codons were mostly favored in the DNA ligase gene of IIV6. The ENC value of 35.64 implied a high CUB in the IIV6 DNA ligase gene. The ENC plot, neutrality plot, parity rule 2 plot, correspondence analysis revealed that mutation pressure and natural selection had an impact on the CUB of the IIVs DNA ligase genes. Additionally, the analysis of codon adaptation index demonstrated that the IIV6 DNA ligase gene is strongly adapted to its host. These findings will improve our comprehension of the CUB of IIV6 DNA ligase and reference genes, which may provide the required information for a fundamental evolutionary analysis of these genes.

Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics.

Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.