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Cold Spring Harbor Protocols Aug 2019DNA ligases are used chiefly to create novel combinations of nucleic acid molecules and to attach them to vectors before molecular cloning. They are either of bacterial...
DNA ligases are used chiefly to create novel combinations of nucleic acid molecules and to attach them to vectors before molecular cloning. They are either of bacterial origin or bacteriophage encoded and have different properties, as discussed here.
Topics: Bacteriophage T4; DNA; DNA Ligases; Enzyme Stability; Escherichia coli; RNA; Temperature
PubMed: 31371476
DOI: 10.1101/pdb.top101352 -
Molecular Cell Nov 2021Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success...
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.
Topics: Animals; BRCA1 Protein; Biopsy; CRISPR-Cas Systems; Cell Line; Cell Nucleus; Cell Proliferation; Chromosome Aberrations; DNA Damage; DNA Ligase ATP; DNA, Single-Stranded; Female; Humans; Lentivirus; MRE11 Homologue Protein; Mammary Neoplasms, Animal; Mice; Mutation; Ovarian Neoplasms; Poly(ADP-ribose) Polymerase Inhibitors; Poly-ADP-Ribose Binding Proteins; RNA, Small Interfering; Transgenes; Triple Negative Breast Neoplasms; Tumor Suppressor p53-Binding Protein 1
PubMed: 34555355
DOI: 10.1016/j.molcel.2021.09.005 -
Experimental & Molecular Medicine Jan 2023Mitochondrial DNA (mtDNA) released through protein oligomers, such as voltage-dependent anion channel 1 (VDAC1), triggers innate immune activation and thus contributes...
Mitochondrial DNA (mtDNA) released through protein oligomers, such as voltage-dependent anion channel 1 (VDAC1), triggers innate immune activation and thus contributes to liver fibrosis. Here, we investigated the role of Parkin, an important regulator of mitochondria, and its regulation of VDAC1-mediated mtDNA release in liver fibrosis. The circulating mitochondrial DNA (mtDNA) and protein levels of liver Parkin and VDAC1 were upregulated in patients with liver fibrosis. A 4-week CCl challenge induced release of mtDNA, activation of STING signaling, a decline in autophagy, and apoptosis in mouse livers, and the knockout of Parkin aggravated these effects. In addition, Parkin reduced mtDNA release and prevented VDAC1 oligomerization in a manner dependent on its E3 activity in hepatocytes. We found that site-specific ubiquitination of VDAC1 at lysine 53 by Parkin interrupted VDAC1 oligomerization and prevented mtDNA release into the cytoplasm under stress. The ubiquitination-defective VDAC1 K53R mutant predominantly formed oligomers that resisted suppression by Parkin. Hepatocytes expressing VDAC1 K53R exhibited mtDNA release and thus activated the STING signaling pathway in hepatic stellate cells, and this effect could not be abolished by Parkin. We propose that the ubiquitination of VDAC1 at a specific site by Parkin confers protection against liver fibrosis by interrupting VDAC1 oligomerization and mtDNA release.
Topics: Mice; Animals; DNA, Mitochondrial; Voltage-Dependent Anion Channel 1; Mitochondria; Ubiquitination; Apoptosis; Ubiquitin-Protein Ligases; Liver Cirrhosis
PubMed: 36658227
DOI: 10.1038/s12276-022-00923-9 -
Nature Oct 2021Transcription-coupled DNA repair removes bulky DNA lesions from the genome and protects cells against ultraviolet (UV) irradiation. Transcription-coupled DNA repair...
Transcription-coupled DNA repair removes bulky DNA lesions from the genome and protects cells against ultraviolet (UV) irradiation. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4 and UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, EC, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4 spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4 lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.
Topics: Carrier Proteins; Cryoelectron Microscopy; DNA Helicases; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Humans; Models, Molecular; Multiprotein Complexes; Poly-ADP-Ribose Binding Proteins; RNA Polymerase II; Transcription Elongation, Genetic; Transcription Factor TFIIH; Transcription Factors; Transcription, Genetic; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34526721
DOI: 10.1038/s41586-021-03906-4 -
Autophagy Nov 2019Macroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the...
Macroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the maintenance of cellular homeostasis, autophagy also involves in regulation of cell cycle progression under nutrient-deprivation conditions. However, whether and how autophagy is regulated by the cell cycle especially during mitosis remains largely undefined. Here we show that WIPI2/ATG18B (WD repeat domain, phosphoinositide interacting 2), an autophagy-related (ATG) protein that plays a critical role in autophagosome biogenesis, is a direct substrate of CUL4-RING ubiquitin ligases (CRL4s). Upon mitosis induction, CRL4s are activated via neddylation, and recruit WIPI2 via DDB1 (damage specific DNA binding protein 1), leading to polyubiquitination and proteasomal degradation of WIPI2 and suppression of autophagy. The WIPI2 protein level and autophagy during mitosis could be rescued by knockdown of or treatment with MLN4924/Pevonedistat, a selective inhibitor of CRLs, via suppression of NAE1 (NEDD8 activating enzyme E1 subunit 1). Moreover, restoration of WIPI2 rescues autophagy during mitosis and leads to mitotic slippage and cell senescence. Our study thus discovers a novel function of CRL4s in autophagy by targeting WIPI2 for polyubiquitination and proteasomal degradation during mitosis. : ACTB, actin beta; ATG, autophagy-related; AMPK, AMP-activated protein kinase; AURKB/ARK2, aurora kinase B; BafA1, bafilomycin A; CCNB1, cyclin B1; CDK1, cyclin dependent kinase 1; CHX, cycloheximide; CQ, chloroquine; CRL4s, CUL4-RING ubiquitin ligases; DDB1, damage specific DNA binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; STK11/LKB1,serine/threonine kinase 11; MTORC1/MTOR complex 1, mechanistic target of rapamycin kinase complex 1; NAE1, NEDD8 activating enzyme E1 subunit 1; NOC, nocodazole; RING, really interesting new gene; RBX1, ring-box 1; SA-GLB1/β-gal, senescence-associated galactosidase beta 1; TSC2, TSC complex subunit 2; TUBA, tubulin alpha; WIPI2, WD repeat domain, phosphoinositide interacting 2.
Topics: Autophagy; Cellular Senescence; Cyclopentanes; DNA-Binding Proteins; HEK293 Cells; HeLa Cells; Humans; Leupeptins; Membrane Proteins; Mitosis; NEDD8 Protein; Phosphate-Binding Proteins; Proteasome Endopeptidase Complex; Protein Binding; Pyrimidines; Signal Transduction; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 30898011
DOI: 10.1080/15548627.2019.1596484 -
DNA Repair Sep 2020To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost... (Review)
Review
To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in DNA replication and repair.
Topics: DNA; DNA Damage; DNA Ligase ATP; DNA Repair; DNA Replication; Humans; Poly-ADP-Ribose Binding Proteins
PubMed: 33087274
DOI: 10.1016/j.dnarep.2020.102908 -
Nature Mar 2024Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis...
Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP). The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.
Topics: Animals; Humans; Mice; Cell Nucleus; Cryoelectron Microscopy; Degrons; DNA Virus Infections; DNA Viruses; DNA, Viral; Immunity, Innate; Innate Immunity Recognition; Interferon Type I; Nuclear Proteins; Nucleosomes; Nucleotidyltransferases; Proteasome Endopeptidase Complex; Protein Stability; Proteolysis; Substrate Specificity; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38418882
DOI: 10.1038/s41586-024-07112-w -
Topics in Current Chemistry (Cham) Mar 2020DNA hydrogels are crosslinked polymeric networks in which DNA is used as the backbone or the crosslinker. These hydrogels are novel biofunctional materials that possess... (Review)
Review
DNA hydrogels are crosslinked polymeric networks in which DNA is used as the backbone or the crosslinker. These hydrogels are novel biofunctional materials that possess the biological character of DNA and the framed structure of hydrogels. Compared with other kinds of hydrogels, DNA hydrogels exhibit not only high mechanical strength and controllable morphologies but also good recognition ability, designable responsiveness, and programmability. The DNA used in this type of hydrogel acts as a building block for self-assembly or as a responsive element due to its sequence recognition ability and switchable structural transitions, respectively. In this review, we describe recent developments in the field of DNA hydrogels and discuss the role played by DNA in these hydrogels. Various synthetic strategies for and a range of applications of DNA hydrogels are detailed.
Topics: DNA; DNA-Directed DNA Polymerase; Hydrogels; Ligases; Light; Nanotechnology; Nucleic Acid Conformation; Nucleic Acid Hybridization
PubMed: 32146604
DOI: 10.1007/s41061-020-0295-7 -
Trends in Cell Biology Apr 2020The cullin-RING E3 ubiquitin ligase CRL4 has emerged as a master regulator of genome stability, which targets key cell cycle proteins for proteolysis during S phase and... (Review)
Review
The cullin-RING E3 ubiquitin ligase CRL4 has emerged as a master regulator of genome stability, which targets key cell cycle proteins for proteolysis during S phase and after DNA damage. Recent advances shed light on how it couples ubiquitination to DNA synthesis, offering a new paradigm for substrate recognition: Cdt2 binds directly onto proliferating cell nuclear antigen (PCNA) loaded on DNA, which serves as a landing pad for the independent recruitment of the ubiquitin ligase and its substrates. Cyclin-dependent kinases (CDKs) and the ataxia telangiectasia and Rad3-related (ATR) kinase ensure accurate spatiotemporal regulation of CRL4 under normal conditions and upon DNA damage. Deregulation of Cdt2 is evident in malignancies and was recently highlighted as a major target of oncogenic viruses, supporting the therapeutic targeting of the ligase as a promising anticancer strategy.
Topics: Animals; Genomic Instability; Humans; Models, Biological; Proteolysis; Substrate Specificity; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 32044173
DOI: 10.1016/j.tcb.2020.01.005 -
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