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Proceedings of the National Academy of... Apr 2024The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1...
The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.
Topics: DNA Replication; Animals; Poly (ADP-Ribose) Polymerase-1; Checkpoint Kinase 1; Mice; Humans; DNA Damage; Ubiquitin-Protein Ligases
PubMed: 38657044
DOI: 10.1073/pnas.2322520121 -
FEBS Letters Oct 2019Successful genome duplication is required for cell proliferation and demands extraordinary precision and accuracy. The mechanisms by which cells enter, progress through,... (Review)
Review
Successful genome duplication is required for cell proliferation and demands extraordinary precision and accuracy. The mechanisms by which cells enter, progress through, and exit S phase are intense areas of focus in the cell cycle and genome stability fields. Key molecular events in the G1 phase of the cell division cycle, especially origin licensing, are essential for pre-establishing conditions for efficient DNA replication during the subsequent S phase. If G1 events are poorly regulated or disordered, then DNA replication can be compromised leading to genome instability, a hallmark of tumorigenesis. Upon entry into S phase, coordinated origin firing and replication progression ensure complete, timely, and precise chromosome replication. Both G1 and S phase progressions are controlled by master cell cycle protein kinases and ubiquitin ligases that govern the activity and abundance of DNA replication factors. In this short review, we describe current understanding and recent developments related to G1 progression and S phase entrance and exit with a particular focus on origin licensing regulation in vertebrates.
Topics: Animals; Carcinogenesis; Cell Cycle; Cell Cycle Checkpoints; Cyclin-Dependent Kinases; DNA Replication; Eukaryotic Cells; G1 Phase; Gene Expression Regulation; Genome; Genomic Instability; Humans; S Phase; Signal Transduction; Ubiquitin-Protein Ligases
PubMed: 31556113
DOI: 10.1002/1873-3468.13619 -
Nature Communications Mar 2024Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the...
Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0-6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.
Topics: Humans; Cryoelectron Microscopy; DNA; Histones; Neoplasms; Nucleosomes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38519511
DOI: 10.1038/s41467-024-46910-8 -
Nucleic Acids Research Jan 2023Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III...
Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.
Topics: Animals; Humans; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Repair; Magnesium; Mammals
PubMed: 36625284
DOI: 10.1093/nar/gkac1263 -
Cell Proliferation Aug 2023Renal ischemia-reperfusion injury (IRI) is mainly responsible for acute kidney injury for which there is no effective therapy. Accumulating evidence has indicated the...
Renal ischemia-reperfusion injury (IRI) is mainly responsible for acute kidney injury for which there is no effective therapy. Accumulating evidence has indicated the important role of mitophagy in mitochondrial homeostasis under stress. OGG1 (8-oxoguanine DNA glycosylase) is known for functions in excision repair of nuclear and mitochondrial DNA. However, the role of OGG1 in renal IRI remains unclear. Herein, we identified OGG1, induced during IRI, as a key factor mediating hypoxia-reoxygenation-induced apoptosis in vitro and renal tissue damage in a renal IRI model. We demonstrated that OGG1 expression during IRI negatively regulates mitophagy by suppressing the PINK1/Parkin pathway, thereby aggravating renal ischemic injury. OGG1 knockout and pharmacological inhibition attenuated renal IRI, in part by activating mitophagy. Our results elucidated the damaging role of OGG1 activation in renal IRI, which is associated with the regulatory role of the PINK1/Parkin pathway in mitophagy.
Topics: Humans; Mitophagy; Reperfusion Injury; Ubiquitin-Protein Ligases; DNA Glycosylases; Protein Kinases
PubMed: 36788635
DOI: 10.1111/cpr.13418 -
Journal of Experimental & Clinical... Apr 2022Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain...
BACKGROUND
Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain challenging to be fully investigated.
METHODS
Genome-wide CRISPR screening combined with transcriptomic sequencing were used to identify the critical genes of doxorubicin resistance. Analysis of clinical samples and datasets, and in vitro and in vivo experiments (including CCK-8, apoptosis, western blot, qRT-PCR and mouse models) were applied to confirm the function of these genes. The bioinformatics and IP-MS assays were utilized to further verify the downstream pathway. RGD peptide-directed and exosome-delivered siRNA were developed for the novel therapy strategy.
RESULTS
We identified that E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin-resistance in OS. Further exploration revealed that Rad18 interact with meiotic recombination 11 (MRE11) to promote the formation of the MRE11-RAD50-NBS1 (MRN) complex, facilitating the activation of the homologous recombination (HR) pathway, which ultimately mediated DNA damage tolerance and leaded to a poor prognosis and chemotherapy response in patients with OS. Rad18-knockout effectively restored the chemotherapy response in vitro and in vivo. Also, RGD-exosome loading chemically modified siRad18 combined with doxorubicin, where exosome and chemical modification guaranteed the stability of siRad18 and the RGD peptide provided prominent targetability, had significantly improved antitumor activity of doxorubicin.
CONCLUSIONS
Collectively, our study identifies Rad18 as a driver of OS doxorubicin resistance that promotes the HR pathway and indicates that targeting Rad18 is an effective approach to overcome chemotherapy resistance in OS.
Topics: Adolescent; Animals; Antibiotics, Antineoplastic; Bone Neoplasms; Clustered Regularly Interspaced Short Palindromic Repeats; DNA-Binding Proteins; Doxorubicin; Humans; Mice; Osteosarcoma; Ubiquitin-Protein Ligases
PubMed: 35459258
DOI: 10.1186/s13046-022-02344-y -
The Journal of Clinical Investigation Mar 2024The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been...
The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.
Topics: Animals; Humans; Mice; Ataxia Telangiectasia Mutated Proteins; B-Lymphocytes; Carcinogenesis; Checkpoint Kinase 1; DNA Replication; Genomic Instability; Mice, Knockout; Nuclear Proteins; Proto-Oncogene Proteins c-myc; Signal Transduction; Sumoylation; Ubiquitin-Protein Ligases
PubMed: 38530355
DOI: 10.1172/JCI167419 -
Molecular Biology Research... Mar 2022Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning...
Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning techniques, as well as molecular study up to now. Despite being a prime cloning tool, DNA ligases still face some shortcomings which lead to their limit of use. Our study provided an improved method that simplified the basic restriction enzyme-based cloning (REC) by eliminating the ligation role, named recombinase-free cloning (RFC). This improved technique was designed with only one PCR reaction, one digestion reaction, and one temperature profile, which takes advantage of endogenous recombinase in host to create the target recombinant vector inside the cell. All purification steps were eliminated for effectively material- and time-saving. Five different clones were generated by RFC. This method showed relatively low efficiency yet successful at a range of 100% in every conducted trial with fragment sizes from 0.5-1.0 kbp. The RFC method could be completed within a day (about 9 hours), without the need of ligase or recombinase or purification steps, which significantly saved DNA components, materials as well as the time required. In conclusion, we expected to provide a more convenient cloning method, as well as enable faster generation of DNA clones, which would be well applied in the less equipped laboratories.
PubMed: 35463820
DOI: 10.22099/mbrc.2021.41923.1685 -
Journal of Virology Mar 2023Type I interferon (IFN-I) response plays a prominent role in innate immunity, which is frequently modulated during viral infection. Here, we report DNA methylation...
Type I interferon (IFN-I) response plays a prominent role in innate immunity, which is frequently modulated during viral infection. Here, we report DNA methylation regulator UHRF1 as a potent negative regulator of IFN-I induction during alphaherpesvirus infection, whereas the viruses in turn regulates the transcriptional expression of UHRF1. Knockdown of UHRF1 in cells significantly increases interferon-β (IFN-β)-mediated gene transcription and viral inhibition against herpes simplex virus 1 (HSV1) and pseudorabies virus (PRV). Mechanistically, UHRF1 deficiency promotes IFN-I production by triggering dsRNA-sensing receptor RIG-I and activating IRF3 phosphorylation. Knockdown of UHRF1 in cells upregulates the accumulation of double-stranded RNA (dsRNA), including host endogenous retroviral sequence (ERV) transcripts, while the treatment of RNase III, known to specifically digest dsRNA, prevents IFN-β induction by siUHRF1. Furthermore, the double-knockdown assay of UHRF1 and DNA methyltransferase DNMT1 suggests that siUHRF1-mediated DNA demethylation may play an important role in dsRNA accumulation and subsequently IFN induction. These findings establish the essential role of UHRF1 in IFN-I-induced antiviral immunity and reveal UHRF1 as a potential antivrial target. Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals, which rely partly on their interaction with IFN-mediated innate immune response. Using alphaherpesviruses PRV and HSV-1 as models, we identified an essential role of DNA methylation regulator UHRF1 in IFN-mediated immunity against virus replication, which unravels a novel mechanism employed by epigenetic factor to control IFN-mediated antiviral immune response and highlight UHRF1, which might be a potential target for antiviral drug development.
Topics: Animals; Humans; Antiviral Agents; CCAAT-Enhancer-Binding Proteins; Gene Expression; Herpesvirus 1, Human; Herpesvirus 1, Suid; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Interferon-beta; Ubiquitin-Protein Ligases; Alphaherpesvirinae; Receptors, Immunologic
PubMed: 36916938
DOI: 10.1128/jvi.00134-23 -
Journal of Translational Medicine Oct 2022DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal...
BACKGROUND
DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM.
METHODS
Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells.
RESULTS
Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo.
CONCLUSION
Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
Topics: Animals; Mice; Annexin A5; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA, Mitochondrial; Flavonoids; Mice, Inbred NOD; Mice, SCID; Multiple Myeloma; Phenols; Recombinant Proteins
PubMed: 36273153
DOI: 10.1186/s12967-022-03705-z