Query：
学者姓名：林忠辉
Refining：
Year
Type
Indexed by
Source
Complex
Co-
Language
Clean All
Abstract ：
Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics. Type III CRISPR systems produce second messengers that activate ancillary nucleases for degradation of viral DNA. Here, structures of one such nuclease, TtCsm6, in various catalytic states reveal how cA4 binding allosterically activates its ribonuclease activity as well as cA4 cleavage for subsequent inactivation.Crystal structures reveal the mechanism of cA4 recognition and cleavage by TtCsm6 cA4 binding in the CARF domain allosterically activates the ribonuclease activity of the HEPN domain. cA4 cleavage by the CARF domain leads to the inactivation of the HEPN ribonuclease. TtCsm6 variants with sustained cA4-activated RNase activity may be applicable in genome editing and diagnostics. Crystal structures show the basis for ancillary nuclease activation and auto-inactivation via second messengers generated in Type III CRISPR systems.
Keyword ：
Allosteric Activation Allosteric Activation Ancillary Nuclease Ancillary Nuclease Csm6 Csm6 Cyclic Oligoadenylate Cyclic Oligoadenylate Type III CRISPR Type III CRISPR
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Du, Liyang , Zhu, Qinwei , Lin, Zhonghui . Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate [J]. | EMBO JOURNAL , 2024 , 43 (2) : 304-315 . |
| MLA | Du, Liyang 等. "Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate" . | EMBO JOURNAL 43 . 2 (2024) : 304-315 . |
| APA | Du, Liyang , Zhu, Qinwei , Lin, Zhonghui . Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate . | EMBO JOURNAL , 2024 , 43 (2) , 304-315 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
Holliday junction (HJ) is a four-way structured DNA intermediate in processes of homologous recombination and DNA double-stranded break (DSB) repair. In bacteria, HJs are processed via either the RuvABC or RecGdependent pathways. In addition, RecG also plays a critical role in the reactivation of stalled replication forks, making it an attractive target for antibacterial drug development. Here, we conducted a high-throughput screening targeting the RecG helicase from a common opportunistic pathogen Pseudomonas aeruginosa (Pa). From a library containing 7920 compounds, we identified Ebselen and TPI-1 (2 ',5 '-Dichloro-[1,1 '-biphenyl]-2,5dione) as two potent PaRecG inhibitors, with IC50 values of 0.31 +/- 0.02 mu M and 1.16 +/- 0.06 mu M, respectively. Further biochemical analyses suggested that both Ebselen and TPI-1 inhibited the ATPase activity of PaRecG, and hindered its binding to HJ DNA with high selectivity. These compounds, when combined with our previously reported RuvAB inhibitors, resulted in more severe DNA repair defects than the individual treatment, and potently enhanced the susceptibility of P. aeruginosa to the DNA damage agents. This work reports novel small molecule inhibitors of RecG, offering valuable chemical tools for advancing our understanding of RecG's function and mechanism. Additionally, these inhibitors might be further developed as promising antibacterial agents in the fight against P. aeruginosa infections.
Keyword ：
Combined effect Combined effect DNA damage repair DNA damage repair Holliday junction Holliday junction Pseudomonas aeruginosa Pseudomonas aeruginosa RecG RecG Small-molecule inhibitors Small-molecule inhibitors
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Li, Longheng , Guo, Binbin , Dai, Lin et al. Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents [J]. | BIOCHEMICAL PHARMACOLOGY , 2024 , 222 . |
| MLA | Li, Longheng et al. "Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents" . | BIOCHEMICAL PHARMACOLOGY 222 (2024) . |
| APA | Li, Longheng , Guo, Binbin , Dai, Lin , Liu, Chun , Lin, Zhonghui . Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents . | BIOCHEMICAL PHARMACOLOGY , 2024 , 222 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation. Graphical Abstract
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Zhang, Danping , Du, Liyang , Gao, Haishan et al. Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system [J]. | NUCLEIC ACIDS RESEARCH , 2024 , 52 (14) : 8419-8430 . |
| MLA | Zhang, Danping et al. "Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system" . | NUCLEIC ACIDS RESEARCH 52 . 14 (2024) : 8419-8430 . |
| APA | Zhang, Danping , Du, Liyang , Gao, Haishan , Yuan, Cai , Lin, Zhonghui . Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system . | NUCLEIC ACIDS RESEARCH , 2024 , 52 (14) , 8419-8430 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase. The precise mechanism of metal-ion catalysis in Holliday junction resolution remains elusive. Here, the authors describe a metal ion-mediated nick and counter-nick mechanism of Holliday junction cleavage by MOC1.
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Zhang, Danping , Xu, Shenjie , Luo, Zhipu et al. MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions [J]. | NATURE COMMUNICATIONS , 2024 , 15 (1) . |
| MLA | Zhang, Danping et al. "MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions" . | NATURE COMMUNICATIONS 15 . 1 (2024) . |
| APA | Zhang, Danping , Xu, Shenjie , Luo, Zhipu , Lin, Zhonghui . MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions . | NATURE COMMUNICATIONS , 2024 , 15 (1) . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
The emergence of chemoresistance poses a significant challenge to the efficacy of DNA-damaging agents in cancer treatment, in part due to the inherent DNA repair capabilities of cancer cells. The Ku70/80 protein complex (Ku) plays a central role in double-strand breaks (DSBs) repair through the classical non-homologous end joining (c-NHEJ) pathway, and has proven to be one of the most promising drug target for cancer treatment when combined with radiotherapy or chemotherapy. In this study, we conducted a high-throughput screening of small-molecule inhibitors targeting the Ku complex by using a fluorescence polarization-based DNA binding assay. From a library of 11,745 small molecules, UMI-77 was identified as a potent Ku inhibitor, with an IC50 value of 2.3 mu M. Surface plasmon resonance and molecular docking analyses revealed that UMI-77 directly bound the inner side of Ku ring, thereby disrupting Ku binding with DNA. In addition, UMI-77 also displayed potent inhibition against MUS81-EME1, a key player in homologous recombination (HR), demonstrating its potential for blocking both NHEJ- and HR-mediated DSB repair pathways. Further cell-based studies showed that UMI-77 could impair bleomycin-induced DNA damage repair, and significantly sensitized multiple cancer cell lines to the DNA-damaging agents. Finally, in a mouse xenograft tumor model, UMI-77 significantly enhanced the chemotherapeutic efficacy of etoposide with little adverse physiological effects. Our work offers a new avenue to combat chemoresistance in cancer treatment, and suggests that UMI-77 could be further developed as a promising candidate in cancer treatment.
Keyword ：
Anticancer Anticancer Chemosensitivity Chemosensitivity DNA repair DNA repair Ku70 Ku70 Ku80 Ku80 NHEJ NHEJ Small-molecule inhibitor Small-molecule inhibitor UMI-77 UMI-77
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Chen, Xuening , Chen, Changkun , Luo, Chengmiao et al. Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo [J]. | EUROPEAN JOURNAL OF PHARMACOLOGY , 2024 , 975 . |
| MLA | Chen, Xuening et al. "Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo" . | EUROPEAN JOURNAL OF PHARMACOLOGY 975 (2024) . |
| APA | Chen, Xuening , Chen, Changkun , Luo, Chengmiao , Liu, Jianyong , Lin, Zhonghui . Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo . | EUROPEAN JOURNAL OF PHARMACOLOGY , 2024 , 975 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC 3.4.22.49), a cysteine protease, plays a critical role in mitosis by cleaving the kleisin subunit of cohesin, thereby presenting a promising target for cancer therapy. However, challenges in isolating active human separase suitable for high-throughput screening have limited the identification of effective inhibitors. Here, we conducted a high-throughput screening of small-molecule inhibitors using the protease domain of Chaetomium thermophilum separase (ctSPD), which not only shares significant sequence similarity with human separase but is also readily available. After conducting a primary screening of a library containing 9,172 compounds and subsequent validation using human separase, we identified walrycin B and its analogs, toxoflavin, 3-methyltoxoflavin, and 3-phenyltoxoflavin, as potent inhibitors of human separase. Subsequent microscale thermophoresis assays and molecular dynamics simulations revealed that walrycin B binds to the active site of separase and competes with substrates for binding. Additionally, cell-based studies showed that walrycin B and its analogs effectively induce cell cycle arrest at the M phase, activate apoptosis, and ultimately lead to cell death in mitosis. Finally, in a mouse xenograft model, walrycin B exhibited significant antitumor efficacy with minimal side effects. Together, these findings highlight the therapeutic potential of walrycin B for cancer treatment and its utility as a chemical tool in future studies involving separase. © 2024 Elsevier Inc.
Keyword ：
Anticancer Anticancer Inhibitor Inhibitor Separase Separase Toxoflavin Toxoflavin Walrycin B Walrycin B
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Zhu, Q. , Du, L. , Wu, J. et al. Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model [J]. | Biochemical Pharmacology , 2024 , 229 . |
| MLA | Zhu, Q. et al. "Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model" . | Biochemical Pharmacology 229 (2024) . |
| APA | Zhu, Q. , Du, L. , Wu, J. , Li, J. , Lin, Z. . Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model . | Biochemical Pharmacology , 2024 , 229 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa (P. aeruginosa, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa. The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal beta-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA.
Keyword ：
branch migration branch migration complex assembly complex assembly DNA damage repair DNA damage repair Holliday junction Holliday junction homologous recombination homologous recombination Pseudomonas aeruginosa Pseudomonas aeruginosa RuvA RuvA RuvB RuvB
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Zhang, Xu , Zhou, Zixuan , Dai, Lin et al. Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from Pseudomonas aeruginosa [J]. | FRONTIERS IN PLANT SCIENCE , 2023 , 14 . |
| MLA | Zhang, Xu et al. "Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from Pseudomonas aeruginosa" . | FRONTIERS IN PLANT SCIENCE 14 (2023) . |
| APA | Zhang, Xu , Zhou, Zixuan , Dai, Lin , Chao, Yulin , Liu, Zheng , Huang, Mingdong et al. Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from Pseudomonas aeruginosa . | FRONTIERS IN PLANT SCIENCE , 2023 , 14 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
The MUS81-EME1/2 structure-specific endonucleases play a crucial role in the processing of stalled replication forks and recombination intermediates, and have been recognized as an attractive drug target to potentiate the anti-cancer efficacy of DNA-damaging agents. Currently, no bioactive small-molecule inhibitors of MUS81 are available. Here, we performed a high-throughput small-molecule inhibitors screening, using the FRET-based DNA cleavage assay. From 7920 compounds, we identified dyngo-4a as a potent inhibitor of MUS81 complexes. Dyngo-4a effectively inhibits the endonuclease activities of both MUS81-EME1 and MUS81-EME2 complexes, with IC50 values of 0.57 mu M and 2.90 & mu;M, respectively. Surface plasmon resonance (SPR) and electrophoretic mobility shift assay (EMSA) assays reveal that dyngo-4a directly binds to MUS81 complexes (KD similar to 0.61 mu M) and prevents them from binding to DNA substrates. In HeLa cells, dyngo-4a significantly suppresses bleomycin-triggered H2AX serine 139 phosphorylation (gamma H2AX). Together, our results demonstrate that dyngo4a is a potent MUS81 inhibitor, which could be further developed as a potentially valuable chemical tool to explore more physiological roles of MUS81 in the cells.
Keyword ：
Dynasore Dynasore Dyngo-4a Dyngo-4a EME1 EME1 EME2 EME2 MUS81 MUS81 Small-molecule inhibitor Small-molecule inhibitor
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Zhang, Xu , Chen, Xuening , Lu, Lian et al. Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening [J]. | BIOORGANIC & MEDICINAL CHEMISTRY , 2023 , 90 . |
| MLA | Zhang, Xu et al. "Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening" . | BIOORGANIC & MEDICINAL CHEMISTRY 90 (2023) . |
| APA | Zhang, Xu , Chen, Xuening , Lu, Lian , Fang, Qianqian , Liu, Chun , Lin, Zhonghui . Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening . | BIOORGANIC & MEDICINAL CHEMISTRY , 2023 , 90 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA(4) in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA(4) recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA(4) reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | Du, Liyang , Zhang, Danping , Luo, Zhipu et al. Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081 [J]. | NUCLEIC ACIDS RESEARCH , 2023 , 51 (5) : 2485-2495 . |
| MLA | Du, Liyang et al. "Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081" . | NUCLEIC ACIDS RESEARCH 51 . 5 (2023) : 2485-2495 . |
| APA | Du, Liyang , Zhang, Danping , Luo, Zhipu , Lin, Zhonghui . Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081 . | NUCLEIC ACIDS RESEARCH , 2023 , 51 (5) , 2485-2495 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Abstract ：
限制-修饰系统(RMS)是细菌为了防御外来DNA入侵而进化产生的一种保护机制.RMS系统可分为Ⅰ型、Ⅱ型、Ⅲ型和Ⅳ型.Asc Ⅰ是一种Ⅱ型限制性核酸内切酶,能识别8-bp DNA基序.尽管Asc Ⅰ已经被广泛应用于分子克隆研究中,然而目前尚无关于Asc Ⅰ蛋白质的表达、纯化以及结构机制等方面的研究报道.本研究基于大肠杆菌重组表达体系建立了 Asc Ⅰ重组蛋白质的高效表达和纯化方法,从每升细菌培养物中可获得约2.5 mg纯度大于95％的Asc Ⅰ蛋白质.进一步的酶学性质研究表明,Asc Ⅰ酶切反应的最适温度是37℃,最适pH为7.5～8.5,反应依赖于Mg2+和Mn2+等二价金属离子.基于小角X射线散射(SAXS)分析技术,我们还建立了AscⅠ蛋白质及其与底物DNA复合物在溶液状态下的三维空间模型,并结合点突变对该模型进行了验证.总之,本研究对Asc Ⅰ蛋白质的重组表达、纯化、酶活性质以及结构机制进行了比较系统地研究,为了解RMS系统的工作机制提供了结构基础,同时也为Asc Ⅰ作为分子克隆工具酶的进一步开发和改造提供了理论依据.
Keyword ：
AscⅠ AscⅠ 小角散射 小角散射 表达纯化 表达纯化 酶学性质 酶学性质 限制性核酸内切酶 限制性核酸内切酶
Cite：
Copy from the list or Export to your reference management。
| GB/T 7714 | 方倩倩 , 陈浩 , 廖馨源 et al. 限制性核酸内切酶Asc Ⅰ蛋白质的表达纯化、酶活测定及小角散射结构解析 [J]. | 中国生物化学与分子生物学报 , 2023 , 39 (2) : 259-268 . |
| MLA | 方倩倩 et al. "限制性核酸内切酶Asc Ⅰ蛋白质的表达纯化、酶活测定及小角散射结构解析" . | 中国生物化学与分子生物学报 39 . 2 (2023) : 259-268 . |
| APA | 方倩倩 , 陈浩 , 廖馨源 , 林忠辉 . 限制性核酸内切酶Asc Ⅰ蛋白质的表达纯化、酶活测定及小角散射结构解析 . | 中国生物化学与分子生物学报 , 2023 , 39 (2) , 259-268 . |
| Export to | NoteExpress RIS BibTex |
Version ：
Export
| Results： |
Selected to |
| Format： |
