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International Journal of Molecular... Jul 2021Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA...
Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in locus and promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.
Topics: DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Fibroblasts; Gene Editing; Genomic Imprinting; HEK293 Cells; Humans; Insulin-Like Growth Factor II; Locus Control Region; Promoter Regions, Genetic; RNA, Long Noncoding; Transcription Factors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; DNA Methyltransferase 3B
PubMed: 34299205
DOI: 10.3390/ijms22147587 -
Molecular Cell Feb 2021Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps...
Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
Topics: Animals; Cell Line, Tumor; Chromatin; DNA Breaks, Single-Stranded; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Female; Humans; Proliferating Cell Nuclear Antigen; Substrate Specificity; Ubiquitin-Protein Ligases; Ubiquitination; Xenopus laevis
PubMed: 33321094
DOI: 10.1016/j.molcel.2020.11.029 -
Cells Mar 2022Plants, as sessile organisms, have limited means to cope with environmental changes. Consequently, they have developed complex regulatory systems to ameliorate abiotic... (Review)
Review
Plants, as sessile organisms, have limited means to cope with environmental changes. Consequently, they have developed complex regulatory systems to ameliorate abiotic stresses im-posed by environmental changes. One such system is the ubiquitin proteasome pathway, which utilizes E3 ligases to target proteins for proteolytic degradation via the 26S proteasome. Plants ex-press a plethora of E3 ligases that are categorized into four major groups depending on their structure. They are involved in many biological and developmental processes in plants, such as DNA repair, photomorphogenesis, phytohormones signaling, and biotic stress. Moreover, many E3 ligase targets are proteins involved in abiotic stress responses, such as salt, drought, heat, and cold. In this review, we will provide a comprehensive overview of E3 ligases and their substrates that have been connected with abiotic stress in order to illustrate the diversity and complexity of how this pathway enables plant survival under stress conditions.
Topics: Gene Expression Regulation, Plant; Plant Proteins; Plants; Stress, Physiological; Ubiquitin-Protein Ligases
PubMed: 35269512
DOI: 10.3390/cells11050890 -
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 -
Frontiers in Plant Science 2021Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to... (Review)
Review
Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.
PubMed: 33897743
DOI: 10.3389/fpls.2021.652170 -
Nucleic Acids Research Jan 2023TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions...
TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.
Topics: Animals; Mice; Humans; DNA Repair; DNA Damage; DNA Replication; DNA-Binding Proteins; Genomics; Fanconi Anemia; Mammals; Ubiquitin-Protein Ligases; Nerve Tissue Proteins
PubMed: 36594163
DOI: 10.1093/nar/gkac1226 -
Nature Communications Jul 2023A challenge in developing proteolysis targeting chimeras (PROTACs) is the establishment of a universal platform applicable in multiple scenarios for precise degradation...
A challenge in developing proteolysis targeting chimeras (PROTACs) is the establishment of a universal platform applicable in multiple scenarios for precise degradation of proteins of interest (POIs). Inspired by the addressability, programmability, and rigidity of DNA frameworks, we develop covalent DNA framework-based PROTACs (DbTACs), which can be synthesized in high-throughput via facile bioorthogonal chemistry and self-assembly. DNA tetrahedra are employed as templates and the spatial position of each atom is defined. Thus, by precisely locating ligands of POI and E3 ligase on the templates, ligand spacings can be controllably manipulated from 8 Å to 57 Å. We show that DbTACs with the optimal linker length between ligands achieve higher degradation rates and enhanced binding affinity. Bispecific DbTACs (bis-DbTACs) with trivalent ligand assembly enable multi-target depletion while maintaining highly selective degradation of protein subtypes. When employing various types of warheads (small molecules, antibodies, and DNA motifs), DbTACs exhibit robust efficacy in degrading diverse targets, including protein kinases and transcription factors located in different cellular compartments. Overall, utilizing modular DNA frameworks to conjugate substrates offers a universal platform that not only provides insight into general degrader design principles but also presents a promising strategy for guiding drug discovery.
Topics: Proteolysis; Ligands; Ubiquitin-Protein Ligases; Ubiquitination; Transcription Factors
PubMed: 37495569
DOI: 10.1038/s41467-023-40244-7 -
Nature Communications Oct 2019DNA methylation, repressive histone marks, and PIWI-interacting RNA (piRNA) are essential for the control of retrotransposon silencing in the mammalian germline....
DNA methylation, repressive histone marks, and PIWI-interacting RNA (piRNA) are essential for the control of retrotransposon silencing in the mammalian germline. However, it remains unknown how these repressive epigenetic pathways crosstalk to ensure retrotransposon silencing in the male germline. Here, we show that UHRF1 is responsible for retrotransposon silencing and cooperates with repressive epigenetic pathways in male germ cells. Conditional loss of UHRF1 in postnatal germ cells causes DNA hypomethylation, upregulation of retrotransposons, the activation of a DNA damage response, and switches in the global chromatin status, leading to complete male sterility. Furthermore, we show that UHRF1 interacts with PRMT5, an arginine methyltransferase, to regulate the repressive histone arginine modifications (H4R3me2s and H3R2me2s), and cooperates with the PIWI pathway during spermatogenesis. Collectively, UHRF1 regulates retrotransposon silencing in male germ cells and provides a molecular link between DNA methylation, histone modification, and the PIWI pathway in the germline.
Topics: Animals; Argonaute Proteins; CCAAT-Enhancer-Binding Proteins; DNA Methylation; Female; Gene Silencing; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Protein Binding; Protein-Arginine N-Methyltransferases; Retroelements; Spermatogenesis; Spermatozoa; Ubiquitin-Protein Ligases
PubMed: 31624244
DOI: 10.1038/s41467-019-12455-4 -
Nucleic Acids Research May 2022Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for...
Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.
Topics: Cell Cycle Proteins; DNA; DNA Breaks, Double-Stranded; Female; HEK293 Cells; Homologous Recombination; Humans; Male; Meiosis; SKP Cullin F-Box Protein Ligases; Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitins
PubMed: 35489071
DOI: 10.1093/nar/gkac304 -
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