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Nature Communications May 2024Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair...
Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.
Topics: Humans; BRCA1 Protein; Ubiquitin-Protein Ligases; Proliferating Cell Nuclear Antigen; DNA Replication; Ubiquitination; Tumor Suppressor Proteins; Phthalazines; Piperazines; Homologous Recombination; Female; HEK293 Cells; Cell Line, Tumor; DNA
PubMed: 38769345
DOI: 10.1038/s41467-024-48427-6 -
BioRxiv : the Preprint Server For... May 2024DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair...
DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair intermediate with 3'-oxidative damage incorporated by DNA polymerase (pol) β at the downstream steps of base excision repair (BER) pathway. Here, we determined X-ray structures of LIG1/nick DNA complexes containing 3'-8oxodG and 3'-8oxorG opposite either a templating Cytosine or Adenine and demonstrated that the ligase active site engages with mutagenic repair intermediates during steps 2 and 3 of the ligation reaction referring to the formation of DNA-AMP intermediate and a final phosphodiester bond, respectively. Furthermore, we showed the mutagenic nick sealing of DNA substrates with 3'-8oxodG:A and 3'-8oxorG:A by LIG1 wild-type, immunodeficiency disease-associated variants, and DNA ligase 3α (LIG3α) . Finally, we observed that LIG1 and LIG3α seal resulting nick after an incorporation of 8oxorGTP:A by polβ and AP-Endonuclease 1 (APE1) can clean oxidatively damaged ends at the final steps. Overall, our findings uncover a mechanistic insight into how LIG1 discriminates DNA or DNA/RNA junctions including oxidative damage and a functional coordination between the downstream enzymes, polβ, APE1, and BER ligases, to process mutagenic repair intermediates to maintain repair efficiency.
PubMed: 38766188
DOI: 10.1101/2024.05.06.592774 -
Acta Biochimica Et Biophysica Sinica Jun 2024Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of...
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 to 10 . 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 . gene, which may be involved in the MMEJ event, and the frequency of MMEJ at . is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ . 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 . 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.
Topics: CRISPR-Cas Systems; Gene Editing; Bacteriophage T7; DNA Ligases; Viral Proteins; Genome, Viral
PubMed: 38761011
DOI: 10.3724/abbs.2024030 -
The EMBO Journal Jun 2024The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC...
The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.
Topics: Decitabine; Humans; DNA (Cytosine-5-)-Methyltransferase 1; Ubiquitin-Protein Ligases; DNA Methylation; Antimetabolites, Antineoplastic; Animals; Sumoylation
PubMed: 38760575
DOI: 10.1038/s44318-024-00108-2 -
Nature Communications May 2024Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the...
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
Topics: Humans; Poly-ADP-Ribose Binding Proteins; Tripartite Motif Proteins; RNA Recognition Motif Proteins; Ubiquitin-Protein Ligases; Stress Granules; RNA Helicases; Ubiquitination; DNA Helicases; Signal Transduction; DEAD Box Protein 58; Transcription Factors; Immunity, Innate; RNA, Double-Stranded; HEK293 Cells; HeLa Cells; Cytoplasmic Granules; RNA Virus Infections; Receptors, Immunologic
PubMed: 38750080
DOI: 10.1038/s41467-024-48596-4 -
Biochemical and Biophysical Research... Aug 2024Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible...
Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible post-translational modification (PTM) in the form of mono- or poly(ADP-ribosyl)ation. This type of modification is known to form a ubiquitin-ADP-ribose (Ub-ADPR) conjugate that depends on the actions of Deltex family of E3 ubiquitin ligases (DTXs). In particular, DTXs add ubiquitin to the 3'-OH of adenosine ribose' in ADP-ribose, which effectively sequesters ubiquitin and impedes ubiquitin-dependent signaling. Previous work demonstrates DTX function for ubiquitination of protein-free ADPR, mono-ADP-ribosylated peptides, and ADP-ribosylated nucleic acids. However, the dynamics of DTX-mediated ubiquitination of poly(ADP-ribosyl)ation remains to be defined. Here we show that the ADPR ubiquitination function is not found in other PAR-binding E3 ligases and is conserved across DTX family members. Importantly, DTXs specifically target poly(ADP-ribose) chains for ubiquitination that can be cleaved by PARG, the primary eraser of poly(ADP-ribose), leaving the adenosine-terminal ADPR unit conjugated to ubiquitin. Our collective results demonstrate the DTXs' specific ubiquitination of the adenosine terminus of poly(ADP-ribosyl)ation and suggest the unique Ub-ADPR conjugation process as a basis for PARP-DTX control of cellular activities.
Topics: Ubiquitin-Protein Ligases; Ubiquitination; Humans; Adenosine Diphosphate Ribose; Poly ADP Ribosylation; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Ubiquitin; ADP-Ribosylation; HEK293 Cells
PubMed: 38749191
DOI: 10.1016/j.bbrc.2024.150101 -
Cell Reports May 2024Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia...
Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia cenocepacia virulence as a cis-2-dodecenoic acid (BDSF) QS signal receptor. Here, we report that the fatty acyl-CoA ligase DsfR (BCAM2136), which efficiently catalyzes in vitro synthesis of lauryl-CoA and oleoyl-CoA from lauric acid and oleic acid, respectively, acts as a global transcriptional regulator to control B. cenocepacia virulence by sensing BDSF. We show that BDSF binds to DsfR with high affinity and enhances the binding of DsfR to the promoter DNA regions of target genes. Furthermore, we demonstrate that the homolog of DsfR in B. lata, RS02960, binds to the target gene promoter, and perception of BDSF enhances the binding activity of RS02960. Together, these results provide insights into the evolved unusual functions of DsfR that control bacterial virulence as a response regulator of QS signal.
Topics: Quorum Sensing; Burkholderia cenocepacia; Virulence; Bacterial Proteins; Coenzyme A Ligases; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Animals; Signal Transduction; Fatty Acids, Monounsaturated; Mice; Protein Binding; Lauric Acids
PubMed: 38748879
DOI: 10.1016/j.celrep.2024.114223 -
Cell Death & Disease May 2024Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key...
Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key pathological contributor to Alzheimer's disease (AD), therefore, the role of Pirh2 was investigated in streptozotocin and oligomer Aβ induced rodent experimental model of AD. Pirh2 protein abundance increased during AD conditions, and transient silencing of Pirh2 inhibited the disease-specific pathological markers like level of p-Tau, βamyloid, acetylcholinesterase activity, and neuronal death. Biochemically, Pirh2 silencing significantly attenuated the oxidative stress, depleted mitochondrial membrane potential, cytochrome c translocation from mitochondria to cytosol, and depleted mitochondrial complex-I activity, and ATP level. Pirh2 silencing also inhibited the altered level of VDAC1, hsp75, hexokinase1, t-Bid, caspase-9, and altered level of apoptotic proteins (Bcl-2, Bax). MALDI-TOF/TOF, co-immunoprecipitation, and UbcH13-linked ubiquitylation assay confirmed the interaction of Pirh2 with cytochrome c and the role of Pirh2 in ubiquitylation of cytochrome c, along with Pirh2-dependent altered proteasome activity. Additionally, Pirh2 silencing further inhibited the translocation of mitochondrion-specific endonuclease G and apoptosis-inducing factors to the nucleus and DNA damage. In conclusion, findings suggested the significant implication of Pirh2 in disease pathogenesis, particularly through impaired mitochondrial function, including biochemical alterations, translocation of cytochrome c, endonuclease G and apoptosis-inducing factor, DNA damage, and neuronal apoptosis.
Topics: Alzheimer Disease; Animals; Cytochromes c; Mitochondria; Neurons; Oxidative Stress; Rats; Male; Ubiquitin-Protein Ligases; Amyloid beta-Peptides; Membrane Potential, Mitochondrial; Ubiquitination; Humans; Apoptosis; Cell Death; Rats, Sprague-Dawley; Disease Models, Animal; Endodeoxyribonucleases
PubMed: 38740775
DOI: 10.1038/s41419-024-06662-1 -
NPJ Breast Cancer May 2024BRCA1 plays a suppressive role in breast tumorigenesis. Ubiquitin-dependent degradation is a common mechanism that regulates BRCA1 protein stability, and several...
BRCA1 plays a suppressive role in breast tumorigenesis. Ubiquitin-dependent degradation is a common mechanism that regulates BRCA1 protein stability, and several ubiquitin ligases involved have been identified. However, the deubiquitinating enzyme for BRCA1 remains less defined. Here, we report that the deubiquitinase USP4 interacts with, deubiquitinates and stabilizes BRCA1, maintaining the protein level of BRCA1. USP4 knockdown results in a decreased BRCA1 protein level, impairment in homologous recombination mediated double-stranded break repair, and increased genome instability, and confers resistance to DNA damage-inducing agents and PARP inhibitors. Ectopic expression of USP4 stabilizes BRCA1 and reverse the effects caused by USP4 knockdown. Moreover, USP4 is low expressed in human breast cancer tissues and its low expression correlates with poorer survival of patients. Furthermore, we identified several loss-of-function mutations of USP4 in human gynecological cancers, the catalytic activity of which or their interaction with BRCA1 is disrupted. Together, we reveal that USP4 is a deubiquitinase for BRCA1. USP4 positively regulates the stability and function of BRCA1 through de-ubiquitination, and plays important role in the suppression of breast cancer.
PubMed: 38734703
DOI: 10.1038/s41523-024-00641-7 -
Biochemistry May 2024The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for...
The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DB1- and UL4-ssociated actors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.
Topics: Humans; DNA-Binding Proteins; Peptides; Fluorescence Polarization; Protein Binding; Ubiquitin-Protein Ligases
PubMed: 38729622
DOI: 10.1021/acs.biochem.4c00044