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Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.The Journal of Allergy and Clinical... Aug 2023Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections,...
BACKGROUND
Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step.
OBJECTIVES
This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance.
METHODS
Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed.
RESULTS
A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4 T cells and low TCR-Vα7.2 T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient.
CONCLUSIONS
This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.
Topics: Humans; DNA Ligases; Autoimmunity; Haploinsufficiency; DNA Ligase ATP; Immunologic Deficiency Syndromes; Mutation; DNA
PubMed: 37004747
DOI: 10.1016/j.jaci.2023.03.022 -
Mutagenesis Feb 2020The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA... (Review)
Review
The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.
Topics: Animals; DNA Damage; DNA Ligase ATP; DNA Repair; Disease; Humans; Immunologic Deficiency Syndromes; Mice; Neoplasms; Poly-ADP-Ribose Binding Proteins
PubMed: 31630206
DOI: 10.1093/mutage/gez026 -
Nucleic Acids Research May 2024Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous...
Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in humans and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-exclusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.
Topics: Animals; Humans; Mice; Cell Line; DNA End-Joining Repair; DNA Ligase ATP; DNA-Activated Protein Kinase; DNA-Binding Proteins; Ku Autoantigen; Translocation, Genetic; V(D)J Recombination; Chromosome Aberrations
PubMed: 38412274
DOI: 10.1093/nar/gkae140 -
Protein and Peptide Letters 2022Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research...
BACKGROUND
Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source.
OBJECTIVE
The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases.
METHODS
We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions.
RESULTS
In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates.
CONCLUSION
Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.
Topics: DNA Ligases; DNA, Recombinant; Escherichia coli; Polyethylene Glycols; Proteins
PubMed: 35657285
DOI: 10.2174/0929866529666220426122432 -
Orphanet Journal of Rare Diseases Oct 2016DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous... (Review)
Review
DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation.Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development.Patients with hypomorphic mutations in LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases.
Topics: DNA Breaks, Double-Stranded; DNA Ligase ATP; DNA Repair; Gene Expression Regulation, Enzymologic; Humans; Immunologic Deficiency Syndromes; Mutation
PubMed: 27717373
DOI: 10.1186/s13023-016-0520-1 -
RNA Biology Aug 2017Over the past 2 decades, different types of circular RNAs have been discovered in all kingdoms of life, and apparently, those circular species are more abundant than... (Review)
Review
Over the past 2 decades, different types of circular RNAs have been discovered in all kingdoms of life, and apparently, those circular species are more abundant than previously thought. Apart from circRNAs in viroids and viruses, circular transcripts have been discovered in rodents more than 20 y ago and recently have been reported to be abundant in many organisms including humans. Their exact function remains still unknown, although one may expect extensive functional studies to follow the currently dominant research into identification and discovery of circRNA by sophisticated sequencing techniques and bioinformatics. Functional studies require models and as such methods for preparation of circRNA in vitro. Here, we will review current protocols for RNA circularization and discuss future prospects in the field.
Topics: Animals; Base Pairing; Computational Biology; Cycloaddition Reaction; DNA Ligases; Exons; Humans; Introns; Nucleic Acid Conformation; RNA; RNA Ligase (ATP); RNA Splicing; RNA, Catalytic; RNA, Circular; RNA, Messenger; Spliceosomes; Viral Proteins
PubMed: 27668458
DOI: 10.1080/15476286.2016.1239009 -
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 -
Structure (London, England : 1993) Aug 2023The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type...
The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.
Topics: Humans; DNA Repair Enzymes; Cryoelectron Microscopy; DNA Repair; DNA End-Joining Repair; DNA Ligase ATP; DNA-Activated Protein Kinase; DNA
PubMed: 37311458
DOI: 10.1016/j.str.2023.05.013 -
Journal of Molecular Biology Jan 2024The joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication,... (Review)
Review
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.
Topics: Animals; Humans; DNA; DNA Damage; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA Replication; X-ray Repair Cross Complementing Protein 1
PubMed: 37714297
DOI: 10.1016/j.jmb.2023.168276 -
Nucleic Acids Research Sep 2020DNA ligases are diverse enzymes with essential functions in replication and repair of DNA; here we review recent advances in their structure and distribution and discuss... (Review)
Review
DNA ligases are diverse enzymes with essential functions in replication and repair of DNA; here we review recent advances in their structure and distribution and discuss how this contributes to understanding their biological roles and technological potential. Recent high-resolution crystal structures of DNA ligases from different organisms, including DNA-bound states and reaction intermediates, have provided considerable insight into their enzymatic mechanism and substrate interactions. All cellular organisms possess at least one DNA ligase, but many species encode multiple forms some of which are modular multifunctional enzymes. New experimental evidence for participation of DNA ligases in pathways with additional DNA modifying enzymes is defining their participation in non-redundant repair processes enabling elucidation of their biological functions. Coupled with identification of a wealth of DNA ligase sequences through genomic data, our increased appreciation of the structural diversity and phylogenetic distribution of DNA ligases has the potential to uncover new biotechnological tools and provide new treatment options for bacterial pathogens.
Topics: Catalysis; DNA Ligases; Genome; Humans; Models, Molecular; Protein Conformation; Structure-Activity Relationship
PubMed: 32365176
DOI: 10.1093/nar/gkaa307