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学者姓名：林忠辉
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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
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| 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 . |
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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
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| 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 et al. "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 . |
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The bacterial restriction-modification system (RMS) plays a pivotal role in cellular defense against foreign DNA. It can be classified into four major groups: types I, II, III and IV. Asc I belongs to the type II restriction endonucleases and can specifically recognize an 8-bp DNA element. Despite being used extensively in molecular cloning, the information for protein expression, purification and three dimensional structure of Asc I has been lacking. Here, we developed a bacterial expression system and an efficient purification procedure, and obtained recombinant Asc I protein with a yield of ~ 2. 5 mg per liter culture for over 95% purity. Further enzymatic characterization showed that the optimum temperature and for enzymatic reaction were 37oC and pH 7. 5~8. 5, respectively. The enzyme required divalent metal ions for activity, with a strong preference for Mg2+ and Mn2+. Furthermore, with the Small Angle X-ray Scattering (SAXS) and extensive mutagenesis analyses, we constructed structural models showing how Asc I might interact with its DNA substrate in solution. Together, our work provides biochemical and structural characterizations of Asc I endonuclease, and paves the way for further applications of the enzyme in molecular cloning. © Editorial Office of Acta Horticulturae Sinica. All rights reserved.
Keyword ：
Asc I Asc I enzymatic characterization enzymatic characterization expression and purification expression and purification restriction endonuclease restriction endonuclease small angle X-ray scattering(SAXS) small angle X-ray scattering(SAXS)
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| GB/T 7714 | Fang, Q.-Q. , Chen, H. , Liao, X.-Y. et al. Expression, Purification and Biochemical and Structural Characterization of the Type II Restriction Endonuclease Asc I; [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]. | Chinese Journal of Biochemistry and Molecular Biology , 2023 , 39 (2) : 259-268 . |
| MLA | Fang, Q.-Q. et al. "Expression, Purification and Biochemical and Structural Characterization of the Type II Restriction Endonuclease Asc I; [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]" . | Chinese Journal of Biochemistry and Molecular Biology 39 . 2 (2023) : 259-268 . |
| APA | Fang, Q.-Q. , Chen, H. , Liao, X.-Y. , Lin, Z.-H. . Expression, Purification and Biochemical and Structural Characterization of the Type II Restriction Endonuclease Asc I; [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] . | Chinese Journal of Biochemistry and Molecular Biology , 2023 , 39 (2) , 259-268 . |
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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
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| 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 . |
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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.
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| 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 . |
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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Mg~(2+)和Mn~(2+)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Asc I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 ：
Asc I Asc I 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
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| 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 (02) : 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 . 02 (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 (02) , 259-268 . |
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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
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| 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 . |
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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
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| 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 . |
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The human MUS81-EME1&2 complexes are structure-selective endonucleases that play important roles in DNA damage repair. Here, we describe a protocol to determine the endonuclease activities of MUS81-EME1&2 complexes toward various DNA structures. We co-express MUS81 with EME1 or EME2 and purify the complexes with high purity, and determine their activities on the cleavages of 3ˊ flaps, 5ˊ flaps, nicked double-stranded DNAs, and Holliday junctions. This protocol can also be used for the determination of substrate preferences of other structure-selective endonucleases. For complete details on the use and execution of this protocol, please refer to Hua et al. (2022). © 2022 The Author(s)
Keyword ：
Molecular Biology Molecular Biology Protein Biochemistry Protein Biochemistry Protein expression and purification Protein expression and purification Structural Biology Structural Biology
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| GB/T 7714 | Fang, Q. , Hua, Z. , Lin, Z. . A protocol to determine the activities of human MUS81-EME1&2 endonucleases [J]. | STAR Protocols , 2022 , 3 (3) . |
| MLA | Fang, Q. et al. "A protocol to determine the activities of human MUS81-EME1&2 endonucleases" . | STAR Protocols 3 . 3 (2022) . |
| APA | Fang, Q. , Hua, Z. , Lin, Z. . A protocol to determine the activities of human MUS81-EME1&2 endonucleases . | STAR Protocols , 2022 , 3 (3) . |
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MUS81 is an important structure-specific endonuclease responsible for the processing of stalled replication forks and recombination intermediates. In human, MUS81 functions by forming complexes with its regulatory subunits EME1 and EME2, playing distinct roles in G2/M and S phases. Although the structures of MUS81-EME1 have been intensively studied, there is no structure information available about MUS81-EME2. Here, we report the crystal structure of MUS81-EME2, which reveals an overall protein fold similar to that of MUS81-EME1 complex. Further biochemical and structural characterization shows that the MUS81-EME1 and MUS81-EME2 complexes are identical in substrate recognition and endonuclease activities in vitro, implying that the distinct cellular roles of the two complexes could arise from temporal controls in cells. Finally, an extensive structure-guided mutagenesis analysis provides implications for the molecular basis of how the MUS81-EME endonucleases recognize various DNA substrates in a structure-selective manner.
Keyword ：
Data and code availability Data and code availability Lead contact Lead contact Materials availability Materials availability
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| GB/T 7714 | Hua, Zhengkang , Fang, Qianqian , Zhang, Danping et al. Crystal structure of the human MUS81-EME2 complex [J]. | STRUCTURE , 2022 , 30 (5) : 743-, . |
| MLA | Hua, Zhengkang et al. "Crystal structure of the human MUS81-EME2 complex" . | STRUCTURE 30 . 5 (2022) : 743-, . |
| APA | Hua, Zhengkang , Fang, Qianqian , Zhang, Danping , Luo, Zhipu , Yuan, Cai , Lin, Zhonghui . Crystal structure of the human MUS81-EME2 complex . | STRUCTURE , 2022 , 30 (5) , 743-, . |
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