208 results on '"Xu Guang Xi"'
Search Results
2. Structural Studies of Pif1 Helicases from Thermophilic Bacteria
- Author
-
Stéphane Réty, Yingzi Zhang, Wentong Fu, Shan Wang, Wei-Fei Chen, and Xu-Guang Xi
- Subjects
Pif1 helicase ,X-ray crystallographic structure ,SAXS ,WYL domain ,molecular modeling ,Biology (General) ,QH301-705.5 - Abstract
Pif1 proteins are DNA helicases belonging to Superfamily 1, with 5′ to 3′ directionality. They are conserved from bacteria to human and have been shown to be particularly important in eukaryotes for replication and nuclear and mitochondrial genome stability. However, Pif1 functions in bacteria are less known. While most Pif1 from mesophilic bacteria consist of the helicase core with limited N-terminal and C-terminal extensions, some Pif1 from thermophilic bacteria exhibit a C-terminal WYL domain. We solved the crystal structures of Pif1 helicase cores from thermophilic bacteria Deferribacter desulfuricans and Sulfurihydrogenibium sp. in apo and nucleotide bound form. We show that the N-terminal part is important for ligand binding. The full-length Pif1 helicase was predicted based on the Alphafold algorithm and the nucleic acid binding on the Pif1 helicase core and the WYL domain was modelled based on known crystallographic structures. The model predicts that amino acids in the domains 1A, WYL, and linker between the Helicase core and WYL are important for nucleic acid binding. Therefore, the N-terminal and C-terminal extensions may be necessary to strengthen the binding of nucleic acid on these Pif1 helicases. This may be an adaptation to thermophilic conditions.
- Published
- 2023
- Full Text
- View/download PDF
3. Remodeling the conformational dynamics of I-motif DNA by helicases in ATP-independent mode at acidic environment
- Author
-
Bo Gao, Ya-Ting Zheng, Ai-Min Su, Bo Sun, Xu-Guang Xi, and Xi-Miao Hou
- Subjects
Biological sciences ,Biochemistry ,Molecular biology ,Structural biology ,Science - Abstract
Summary: I-motifs are noncanonical four-stranded DNA structures formed by C-rich sequences at acidic environment with critical biofunctions. The particular pH sensitivity has inspired the development of i-motifs as pH sensors and DNA motors in nanotechnology. However, the folding and regulation mechanisms of i-motifs remain elusive. Here, using single-molecule FRET, we first show that i-motifs are more dynamic than G4s. Impressively, i-motifs display a high diversity of six folding species with slow interconversion. Further results indicate that i-motifs can be linearized by Replication protein A. More importantly, we identified a number of helicases with high specificity to i-motifs at low pH. All these helicases directly act on and efficiently resolve i-motifs into intermediates independent of ATP, although they poorly unwind G4 or duplex at low pH. Owing to the extreme sensitivity to helicases and no need for ATP, i-motif may be applied as a probe for helicase sensing both in vitro and in vivo.
- Published
- 2022
- Full Text
- View/download PDF
4. Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA
- Author
-
Yi-Ran Wang, Ting-Ting Guo, Ya-Ting Zheng, Chang-Wei Lai, Bo Sun, Xu-Guang Xi, and Xi-Miao Hou
- Subjects
Molecular structure ,Molecular biology ,Science - Abstract
Summary: G-quadruplexes (G4s) are non-canonical DNA structures with critical roles in DNA metabolisms. To resolve those structures that can cause replication fork stalling and genomic instability, single-stranded DNA-binding proteins and helicases are required. Here, we characterized the interplay between RPA and helicases on G4s using single-molecule FRET. We first discovered that human RPA efficiently prevents G4 formation by preempting ssDNA before its folding. RPA also differentially interacts with the folded G4s. However, helicases such as human BLM and yeast Pif1 have different G4 preferences from RPA mainly based on loop lengths. More importantly, both RPA and these helicases are required for the stable G4 unfolding, as RPA promotes helicase-mediated repetitive unfolding into durative linear state. Furthermore, BLM can traverse G4 obstacles temporarily disrupted by RPA and continue to unwind downstream duplex. We finally proposed the mechanisms underlying above functions of RPA in preventing, resolving, and assisting helicases to eliminate G4s.
- Published
- 2021
- Full Text
- View/download PDF
5. Construction, expression, and characterization of AG11–843 and AG11–1581
- Author
-
Xie Yan, Yan-Tao Yang, Wei Shi, Xia Ai, and Xu-Guang Xi
- Subjects
Computer applications to medicine. Medical informatics ,R858-859.7 ,Science (General) ,Q1-390 - Abstract
This data article contains descriptive and experimental data on the construction, expression, and simple characterization of AG11–843 and AG11–1581. AG1 is an important member of the DUF1220 protein family. It׳s hard to get the recombinant protein because of its DNA sequence. The DNA sequence were optimized by proper design, cloned by overlap PCR and constructed into expression vector. AG11–843 and AG11–1581.were over expressed in Escherichia coli, purified and analyzed by dynamic light scattering and gel filtration analysis. An effective technique is provided to construct and express proteins with complicated sequences. Keywords: Gene synthesis, Plasmid construction, PCR, Recombinant protein expression, DUF1220, AG1
- Published
- 2018
- Full Text
- View/download PDF
6. A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay
- Author
-
Fang-Yuan Teng, Zong-Zhe Jiang, Ling-Yun Huang, Man Guo, Feng Chen, Xi-Miao Hou, Xu-Guang Xi, and Yong Xu
- Subjects
fluorescence ,molecular interaction ,molecular dynamic ,DNA repair ,single molecule ,helicase ,Biology (General) ,QH301-705.5 - Abstract
Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the Escherichia coli RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3′ and 5′ ends of the overhang DNA dynamically in vitro for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.
- Published
- 2020
- Full Text
- View/download PDF
7. Human RPA activates BLM’s bidirectional DNA unwinding from a nick
- Author
-
Zhenheng Qin, Lulu Bi, Xi-Miao Hou, Siqi Zhang, Xia Zhang, Ying Lu, Ming Li, Mauro Modesti, Xu-Guang Xi, and Bo Sun
- Subjects
BLM ,RPA ,helicase ,single molecule ,optical tweezers ,DNA unwinding ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.
- Published
- 2020
- Full Text
- View/download PDF
8. Bioorthogonal dissociative rhenium(I) photosensitisers for controlled immunogenic cell death induction.
- Author
-
Xu, Guang-Xi, Lee, Lawrence Cho-Cheung, Leung, Peter Kam-Keung, Mak, Eunice Chiu-Lam, Shum, Justin, Zhang, Kenneth Yin, Zhao, Qiang, and Lo, Kenneth Kam-Wing
- Published
- 2023
- Full Text
- View/download PDF
9. Folding Kinetics of Single Human Telomeric G‑Quadruplex Affected by Cisplatin
- Author
-
Hai-Peng Ju, Yi-Zhou Wang, Jing You, Xi-Miao Hou, Xu-Guang Xi, Shuo-Xing Dou, Wei Li, and Peng-Ye Wang
- Subjects
Chemistry ,QD1-999 - Published
- 2016
- Full Text
- View/download PDF
10. Biochemical and functional characterization of an exonuclease from Chaetomium thermophilum
- Author
-
Ling-Gang Yuan, Na-Nv Liu, and Xu-Guang Xi
- Subjects
Exonucleases ,Exodeoxyribonucleases ,Werner Syndrome Helicase ,RecQ Helicases ,Biophysics ,Cell Biology ,Chaetomium ,Molecular Biology ,Biochemistry - Abstract
Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ
- Published
- 2022
11. G-quadruplex DNA: a novel target for drug design
- Author
-
Feng Chen, Man Guo, Xu-Guang Xi, Zongzhe Jiang, Fang-Yuan Teng, Yong Xu, and Xiaozhen Tan
- Subjects
DNA Replication ,Genome instability ,Transcription, Genetic ,Computational biology ,Biology ,G-quadruplex ,Telomestatin ,DNA sequencing ,Epigenesis, Genetic ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Animals ,Humans ,Epigenetics ,Molecular Biology ,Pharmacology ,DNA replication ,Helicase ,DNA ,Cell Biology ,Telomere ,G-Quadruplexes ,chemistry ,Drug Design ,biology.protein ,Molecular Medicine - Abstract
G-quadruplex (G4) DNA is a type of quadruple helix structure formed by a continuous guanine-rich DNA sequence. Emerging evidence in recent years authenticated that G4 DNA structures exist both in cell-free and cellular systems, and function in different diseases, especially in various cancers, aging, neurological diseases, and have been considered novel promising targets for drug design. In this review, we summarize the detection method and the structure of G4, highlighting some non-canonical G4 DNA structures, such as G4 with a bulge, a vacancy, or a hairpin. Subsequently, the functions of G4 DNA in physiological processes are discussed, especially their regulation of DNA replication, transcription of disease-related genes (c-MYC, BCL-2, KRAS, c-KIT et al.), telomere maintenance, and epigenetic regulation. Typical G4 ligands that target promoters and telomeres for drug design are also reviewed, including ellipticine derivatives, quinoxaline analogs, telomestatin analogs, berberine derivatives, and CX-5461, which is currently in advanced phase I/II clinical trials for patients with hematologic cancer and BRCA1/2-deficient tumors. Furthermore, since the long-term stable existence of G4 DNA structures could result in genomic instability, we summarized the G4 unfolding mechanisms emerged recently by multiple G4-specific DNA helicases, such as Pif1, RecQ family helicases, FANCJ, and DHX36. This review aims to present a general overview of the field of G-quadruplex DNA that has progressed in recent years and provides potential strategies for drug design and disease treatment.
- Published
- 2021
12. DEAD-box RNA helicase Dbp2 binds to G-quadruplex nucleic acids and regulates different conformation of G-quadruplex DNA
- Author
-
Qin-Xia Song, Chang-Wei Lai, Na-Nv Liu, Xi-Miao Hou, and Xu-Guang Xi
- Subjects
DEAD-box RNA Helicases ,G-Quadruplexes ,Saccharomyces cerevisiae Proteins ,Biophysics ,Humans ,Cell Biology ,DNA ,Saccharomyces cerevisiae ,Molecular Biology ,Biochemistry - Abstract
G-quadruplexes (G4s) are important in regulating DNA replication, repair and RNA transcription through interactions with specialized proteins. Dbp2 has been identified as a G4 DNA binding protein from Saccharomyces cerevisiae cell lysates. The majority of G4 motifs in Saccharomyces cerevisiae display 5-50 nt loops, only a few have 1-2 nt loops. Human DDX5 could unfold MycG4 DNA, whether Dbp2 also participates in remodeling G4 motifs with short loops in Saccharomyces cerevisiae remains elusive. Here we find that Dbp2 prefers G-rich substrates and binds MycG4 with a high affinity. Dbp2 possesses a dual function for different conformations of MycG4, destabilizing the folded MycG4 and inducing further folding of the unfolded MycG4. Similarly, DDX5 can unfold MycG4, but it exhibits a weaker MycG4 folding-promoting activity relative to Dbp2. Furthermore, Dbp2 facilitates DNA annealing activity in the absence of ATP, suggesting that Dbp2 can work on DNA substrates and possibly participate in DNA metabolism. Our results demonstrate that Dbp2 plays an important role in regulating the folding and unfolding activities of MycG4.
- Published
- 2022
13. Bloom Syndrome Helicase Compresses Single‐Stranded DNA into Phase‐Separated Condensates
- Author
-
Teng Wang, Jiaojiao Hu, Yanan Li, Lulu Bi, Lijuan Guo, Xinshuo Jia, Xia Zhang, Dan Li, Xi‐Miao Hou, Mauro Modesti, Xu‐Guang Xi, Cong Liu, Bo Sun, ShanghaiTech University [Shanghai], Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences [Beijing] (CAS), Shanghai Jiao Tong University [Shanghai], Northwest A and F University, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Modesti, Mauro, Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)
- Subjects
DNA Repair ,RecQ Helicases ,Single-Molecule Studies ,[SDV]Life Sciences [q-bio] ,Biomolecular Condensate ,DNA, Single-Stranded ,DNA ,General Chemistry ,General Medicine ,Genomic Instability ,Catalysis ,Bloom Syndrome Protein ,Helicase ,[SDV] Life Sciences [q-bio] ,Phase Separation ,Adenosine Triphosphate ,Replication Protein A ,Humans ,Bloom Syndrome - Abstract
International audience; Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.
- Published
- 2022
14. Targeting the RNA G-Quadruplex and Protein Interactome for Antiviral Therapy
- Author
-
Li-Yan Zhai, Jing-Fan Liu, Jian-Jin Zhao, Ai-Min Su, Xu-Guang Xi, and Xi-Miao Hou
- Subjects
G-Quadruplexes ,Epstein-Barr Virus Infections ,Herpesvirus 4, Human ,Drug Discovery ,Molecular Medicine ,Humans ,RNA ,Ligands ,Antiviral Agents - Abstract
In recent years, G-quadruplexes (G4s), types of noncanonical four-stranded nucleic acid structures, have been identified in many viruses that threaten human health, such as HIV and Epstein-Barr virus. In this context, G4 ligands were designed to target the G4 structures, among which some have shown promising antiviral effects. In this Perspective, we first summarize the diversified roles of RNA G4s in different viruses. Next, we introduce small-molecule ligands developed as G4 modulators and highlight their applications in antiviral studies. In addition to G4s, we comprehensively review the medical intervention of G4-interacting proteins from both the virus (N protein, viral-encoded helicases, severe acute respiratory syndrome-unique domain, and Epstein-Barr nuclear antigen 1) and the host (heterogeneous nuclear ribonucleoproteins, RNA helicases, zinc-finger cellular nucelic acid-binding protein, and nucleolin) by inhibitors as an alternative way to disturb the normal functions of G4s. Finally, we discuss the challenges and opportunities in G4-based antiviral therapy.
- Published
- 2022
15. Nonstructural N- and C-tails of Dbp2 confer the protein full helicase activities
- Author
-
Qin-Xia Song, Na-Nv Liu, Zhao-Xia Liu, Ying-Zi Zhang, Stephane Rety, Xi-Miao Hou, Xu-Guang Xi, Northwest A and F University, Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biotechnologie et Pharmacogénétique Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), and National Natural Science Foundation of China (32071291,32201042, 32071225, and 31870788)CNRS LIA ('Helicase-mediated G-quadruplex DNA unwinding and genome stability').
- Subjects
disordered protein regions ,RNA-protein interaction ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,RNA helicase Dbp2 ,FRET ,Cell Biology ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,RNA helicase Dbp2 disordered protein regions RNA-protein interaction X-ray crystallography FRET ,Molecular Biology ,Biochemistry ,X-ray crystallography - Abstract
International audience; Human DDX5 and its yeast ortholog Dbp2 are ATP-dependent RNA helicases that play a keyrole in normal cell processes, cancer development and viral infection. The crystal structure ofthe RecA1-like domain of DDX5 is available, but the global structure of DDX5/Dbp2subfamily proteins remains to be elucidated. Here, we report the first X-ray crystal structuresof the Dbp2 helicase core alone and in complex with adenosine diphosphate nucleotide (ADP)at 3.22 Å and 3.05 Å resolutions, respectively. The structures of the ADP-bound post-hydrolysisstate and apo-state demonstrate the conformational changes that occur when the nucleotides arereleased. Our results showed that the helicase core of Dbp2 shifted between open and closedconformation in solution, but the unwinding activity was hindered when the helicase core wasrestricted to a single conformation. A small-angle X-ray scattering (SAXS) experiment showedthat the disordered amino- (N-) and carboxy- (C-) tails are flexible in solution. Truncationmutations confirmed that the N- and C-tails were critical for the nucleic acid binding, ATPase,and unwinding activities, with the C-tail being exclusively responsible for the annealing activity.Furthermore, we labeled the terminal tails to observe the conformational changes between thedisordered tails and the helicase core upon binding nucleic acid substrates. Specifically, wefound that the nonstructural N- and C-tails bind to RNA substrates and tether them to thehelicase core domain, thereby conferring full helicase activities to the Dbp2 protein. Thisdistinct structural characteristic provides new insight into the mechanism of DEAD-box RNAhelicases.
- Published
- 2023
16. The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition
- Author
-
Lulu Bi, Zhenheng Qin, Teng Wang, Yanan Li, Xinshuo Jia, Xia Zhang, Xi-Miao Hou, Mauro Modesti, Xu-Guang Xi, Bo Sun, ShanghaiTech University [Shanghai], Shanghai Institute of Biochemistry and Cell Biology [Shanghai, China], University of Chinese Academy of Sciences [Beijing] (UCAS), Northwest A and F University, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), and Modesti, Mauro
- Subjects
Microscopy, Confocal ,Multidisciplinary ,Optical Tweezers ,RecQ Helicases ,[SDV]Life Sciences [q-bio] ,DNA, Single-Stranded ,DNA ,single molecule ,oligomerization ,[SDV] Life Sciences [q-bio] ,helicase ,Homologous Recombination ,SSB ,BLM - Abstract
International audience; Helicases are multifunctional motor proteins with the primary task of separating nucleic acid duplexes. These enzymes often exist in distinct oligomeric forms and play essential roles during nucleic acid metabolism. Whether there is a correlation between their oligomeric state and cellular function, and how helicases effectively perform functional switching remains enigmatic. Here, we address these questions using a combined single-molecule approach and Bloom syndrome helicase (BLM). By examining the head-on collision of two BLM-mediated DNA unwinding forks, we find that two groups of BLM, upon fork convergence, promptly oligomerize across the fork junctions and tightly bridge two independent single-stranded (ss) DNA molecules that were newly generated by the unwinding BLMs. This protein oligomerization is mediated by the helicase and RNase D C-terminal (HRDC) domain of BLM and can sustain a disruptive force of up to 300 pN. Strikingly, onsite BLM oligomerization gives rise to an immediate transition of their helicase activities, from unwinding dsDNA to translocating along ssDNA at exceedingly fast rates, thus allowing for the efficient displacement of ssDNA-binding proteins, such as RPA and RAD51. These findings uncover an activity transition pathway for helicases and help to explain how BLM plays both pro- and anti-recombination roles in the maintenance of genome stability.
- Published
- 2022
17. Macromolecular aging: ATP hydrolysis-driven functional and structural changes in Escherichia coli RecQ helicase
- Author
-
Hou-Qiang Xu, Xu-Guang Xi, and Dan Li
- Subjects
0301 basic medicine ,Chemistry ,RecQ helicase ,Biophysics ,Cell Biology ,medicine.disease_cause ,Biochemistry ,Structure and function ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,ATP hydrolysis ,030220 oncology & carcinogenesis ,medicine ,Molecular Biology ,Escherichia coli ,Macromolecule - Abstract
Aging has been considered a phenomenon that can be only applied to cells or organisms. Here, we show that RecQ helicase from E. coli displays an aging phenomenon: this macromolecular motor loses its structure and function after hydrolyzing a certain number of ATP molecules. The aging process was only triggered by repeated catalytic cycles. These observations lead to a new concept: macromolecule aging.
- Published
- 2021
18. Structural mechanism underpinning Thermus oshimai Pif1‐mediated G‐quadruplex unfolding
- Author
-
Yang‐Xue Dai, Hai‐Lei Guo, Na‐Nv Liu, Wei‐Fei Chen, Xia Ai, Hai‐Hong Li, Bo Sun, Xi‐Miao Hou, Stephane Rety, Xu‐Guang Xi, Northwest A and F University, ShanghaiTech University [Shanghai], Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), RETY, Stephane, and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
- Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,DNA Helicases ,Articles ,DNA ,G4-Recognizing Surface ,Biochemistry ,G-quadruplexes ,structures ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Genomic Instability ,X-ray ,Genetics ,Humans ,ToPif1 ,Thermus ,[SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Molecular Biology - Abstract
International audience; G-quadruplexes (G4s) are unusual stable DNA structures that cause genomic instability. To overcome the potential barriers formed by G4s, cells have evolved different families of proteins that unfold G4s. Pif1 is a DNA helicase from superfamily 1 (SF1) conserved from bacteria to humans with high G4-unwinding activity. Here, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif1) complexed with a G4. Our structure reveals that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B which constitute a G4-Recognizing Surface (GRS). The overall structure of the G4 maintains its three-layered propeller-type G4 topology, without significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 are recognized by GRS residues mainly through electrostatic, ionic interactions, and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Compared with previously solved structures of SF2 helicases in complex with G4, our structure reveals how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.
- Published
- 2022
19. Photofunctional cyclometallated iridium(iii) polypyridine methylsulfone complexes as sulfhydryl-specific reagents for bioconjugation, bioimaging and photocytotoxic applications
- Author
-
Huang, Lili, primary, Leung, Peter Kam-Keung, additional, Lee, Lawrence Cho-Cheung, additional, Xu, Guang-Xi, additional, Lam, Yun-Wah, additional, and Lo, Kenneth Kam-Wing, additional
- Published
- 2022
- Full Text
- View/download PDF
20. Photo‐ and Electrochemical Dual‐Responsive Iridium Probe for Saccharide Detection
- Author
-
Carrod, Andrew J., primary, Graglia, Francesco, additional, Male, Louise, additional, Le Duff, Cécile, additional, Simpson, Peter, additional, Elsherif, Mohamed, additional, Ahmed, Zubair, additional, Butt, Haider, additional, Xu, Guang‐Xi, additional, Kam‐Wing Lo, Kenneth, additional, Bertoncello, Paolo, additional, and Pikramenou, Zoe, additional
- Published
- 2021
- Full Text
- View/download PDF
21. Structural study of the function of Candida Albicans Pif1
- Author
-
Stéphane Réty, Na-Nv Liu, Xu-Guang Xi, Ben-Ge Xin, Dan Li, Teng Zhang, Ke-Yu Lu, Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
- Subjects
0301 basic medicine ,Models, Molecular ,Conformational change ,Protein Conformation ,[SDV]Life Sciences [q-bio] ,Saccharomyces cerevisiae ,Mutant ,Biophysics ,DNA, Single-Stranded ,Mitochondrion ,Crystallography, X-Ray ,Biochemistry ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Adenosine Triphosphate ,Candida albicans ,Humans ,Molecular Biology ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Candidiasis ,DNA Helicases ,Helicase ,Cell Biology ,DNA ,biology.organism_classification ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Thymidine - Abstract
International audience; Pif1 helicases, conserved in eukaryotes, are involved in maintaining genome stability in both the nucleus and mitochondria. Here, we report the crystal structure of a truncated Candida Albicans Pif1 (CaP-if1 368À883) in complex with ssDNA and an ATP analog. Our results show that the Q-motif is responsible for identifying adenine bases, and CaPif1 preferentially utilizes ATP/dATP during dsDNA unwinding. Although CaPif1 shares structural similarities with Saccharomyces cerevisiae Pif1, CaPif1 can contact the thymidine bases of DNA by hydrogen bonds, whereas ScPif1 cannot. More importantly, the crosslinking and mutant experiments have demonstrated that the conformational change of domain 2B is necessary for CaPif1 to unwind dsDNA. These findings contribute to further the understanding of the unwinding mechanism of Pif1.
- Published
- 2021
22. Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi
- Author
-
Ze-Yu Song, Daniel Auguin, Bo Sun, Wei-Fei Chen, Xu-Guang Xi, Shuo-Xing Dou, Liu-Tao Zhai, Stéphane Réty, Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Ecole des Hautes Etudes Commerciales (HEC Paris), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), Université d'Orléans (UO)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ShanghaiTech University [Shanghai], Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP), University of Chinese Academy of Sciences [Beijing] (UCAS), Northwest A and F University, xi, Xu-Guang, Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)
- Subjects
Models, Molecular ,Telomerase ,AcademicSubjects/SCI00010 ,[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Amino Acid Motifs ,In Vitro Techniques ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Catalysis ,03 medical and health sciences ,Structural Biology ,Catalytic Domain ,Candida albicans ,Escherichia coli ,Genetics ,medicine ,Telomerase reverse transcriptase ,Candida tropicalis ,Structural motif ,030304 developmental biology ,0303 health sciences ,Mutation ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,030302 biochemistry & molecular biology ,RNA ,medicine.disease ,Dynamic Light Scattering ,Recombinant Proteins ,Reverse transcriptase ,Telomere ,Chronic traumatic encephalopathy ,Biochemistry ,Chromatography, Gel - Abstract
Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.
- Published
- 2021
23. Utilization of Rhenium(I) Polypyridine Complexes Featuring a Dinitrophenylsulfonamide Moiety as Biothiol‐Selective Phosphorogenic Bioimaging Reagents and Photocytotoxic Agents
- Author
-
Xu, Guang‐Xi, primary, Lee, Lawrence Cho‐Cheung, additional, Kwok, Cyrus Wing‐Ching, additional, Leung, Peter Kam‐Keung, additional, Zhu, Jing‐Hui, additional, and Lo, Kenneth Kam‐Wing, additional
- Published
- 2021
- Full Text
- View/download PDF
24. Endogenous Bos taurus RECQL is predominantly monomeric and more active than oligomers
- Author
-
Hu Yin, Ben-Ge Xin, Stéphane Réty, Ze-Yu Song, Wei-Fei Chen, Shuo-Xing Dou, Lei Ji, Hai-Lei Guo, Na-Nv Liu, Qing-Man Wang, Xu-Guang Xi, Yang-Xue Dai, Xi-Miao Hou, Xia Ai, Northwest A and F University, University of Chinese Academy of Sciences [Beijing] (UCAS), Laboratoire de biologie et pharmacologie appliquée (LBPA), and Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay)
- Subjects
ATPase ,Dimer ,Breast Neoplasms ,Oligomer ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,law ,Holliday junction ,Animals ,Genetic Predisposition to Disease ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,030304 developmental biology ,0303 health sciences ,RecQ Helicases ,biology ,030302 biochemistry & molecular biology ,Helicase ,DNA ,Recombinant Proteins ,G-Quadruplexes ,Monomer ,chemistry ,Biochemistry ,biology.protein ,Recombinant DNA ,Cattle - Abstract
International audience; There is broad consensus that RecQ family helicase is a high-order oligomer that dissociates into a dimer upon ATP binding. This conclusion is based mainly on studies of highly purified recombinant proteins, and the oligomeric states of RecQ helicases in living cells remain unknown. We show here that, in contrast to current models, monomeric RECQL helicase is more abundant than oligomer/dimer forms in living cells. Further characterization of endogenous BtRECQL and isolated monomeric BtRECQL using various approaches demonstrates that both endogenous and recombinant monomeric BtRECQL effectively function as monomers, displaying higher helicase and ATPase activities than dimers and oligomers. Furthermore, monomeric BtRECQL unfolds intramolecular G-quadruplex DNA as efficiently as human RECQL and BLM helicases. These discoveries have implications for understanding endogenous RECQL oligomeric structures and their regulation. It is worth revisiting oligomeric states of the other members of the RecQ family helicases in living cells.
- Published
- 2021
25. Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition
- Author
-
Xu-Guang Xi, Wei-Fei Chen, Hai-Lei Guo, Fang-Yuan Teng, Shuo-Xing Dou, Yang-Xue Dai, Na-Nv Liu, Xi-Miao Hou, Stéphane Réty, Northwest A and F University, University of Chinese Academy of Sciences [Beijing] (UCAS), Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and xi, Xu-Guang
- Subjects
Models, Molecular ,AcademicSubjects/SCI00010 ,Protein Conformation ,[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Dimer ,DNA, Single-Stranded ,Plasma protein binding ,Biology ,Mitochondrion ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Bacterial Proteins ,Structural Biology ,Genetics ,medicine ,Thermus ,030304 developmental biology ,0303 health sciences ,Molecular Structure ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,030302 biochemistry & molecular biology ,DNA Helicases ,Helicase ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,medicine.anatomical_structure ,chemistry ,Duplex (building) ,Biophysics ,biology.protein ,Protein Multimerization ,[SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Nucleus ,DNA ,Protein Binding - Abstract
Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.
- Published
- 2021
26. DHX36-mediated G-quadruplex unfolding is ATP-independent?
- Author
-
Hai-Lei Guo, Na-Nv Liu, Shuo-Xing Dou, Stephane Rety, Wei-Fei Chen, Xi-Miao Hou, Xu-Guang Xi, Yan-Xue Dai, Ze-Yu Song, Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)
- Subjects
Quantitative Biology - Biomolecules ,Biological Physics (physics.bio-ph) ,FOS: Biological sciences ,[SDV]Life Sciences [q-bio] ,FOS: Physical sciences ,Biomolecules (q-bio.BM) ,Physics - Biological Physics - Abstract
Chen et al. solved the crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex (G4) and a single-stranded DNA segment. They believed that the mechanism they proposed may represent a general model for describing how a G4-unfolding helicase recognizes and unfolds G4 DNA. Their conclusion is interesting, however, we noticed that their linear DNA substrate (DNAMyc) that harbors a Myc-promoter-derived G4-forming sequence was directly used without pre-folding. This raises the question whether the structure they obtained really reflects DHX36-mediated G4 recognition and unfolding, or just only represents a DHX36-binding-induced quasi-folded G4 structure. By a combination of polymerase extension, DMS footprinting, stopped-flow, and smFRET assays, we obtained clear evidences that do not support their ATP-independent one-base translocation structural model. We further revealed that the oscillation of FRET signal they observed should correspond to a repetitive G4 binding, but not unfolding, by DHX36.
- Published
- 2020
27. Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays
- Author
-
Qian Guo, Xiao-Mei Li, Lei Ji, Wen-Qiang Wu, Xu-Guang Xi, Yang-Xue Dai, Na-Nv Liu, Hai-Lei Guo, Ke-Yu Lu, Dept. of Electrical and Information Technology, Lund University, Lund University [Lund], Modèles de Cellules Souches Malignes et Thérapeutiques, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de biologie et pharmacologie appliquée (LBPA), and Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay)
- Subjects
Dna duplex ,Chromosome replication ,[SDV]Life Sciences [q-bio] ,02 engineering and technology ,G-quadruplex ,01 natural sciences ,Biochemistry ,Fluorescence ,Substrate Specificity ,Analytical Chemistry ,DEAD-box RNA Helicases ,Animals ,Drosophila Proteins ,Humans ,ComputingMilieux_MISCELLANEOUS ,Enzyme Assays ,RecQ Helicases ,biology ,Chemistry ,010401 analytical chemistry ,Helicase ,DNA ,021001 nanoscience & nanotechnology ,Stopped flow ,Enzymes ,0104 chemical sciences ,G-Quadruplexes ,Kinetics ,Spectrometry, Fluorescence ,Intramolecular force ,Bioanalytical methods ,biology.protein ,Biophysics ,Drosophila ,0210 nano-technology - Abstract
International audience; G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s −1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicasemediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets.
- Published
- 2020
28. The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex
- Author
-
Fang-Yuan Teng, Ting-Ting Wang, Xu-Guang Xi, Hai-Lei Guo, Xi-Miao Hou, Ben-Ge Xin, Shuo-Xing Dou, Bo Sun, Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Department of Therapeutic Radiology, Yale University [New Haven], Laboratory of Soft Matter Physics, and Institute of Physics, Chinese Academy of Sciences
- Subjects
0301 basic medicine ,Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA repair ,RecQ helicase ,Unwinding ,[SDV]Life Sciences [q-bio] ,Protein domain ,G-quadruplex ,Biochemistry ,Substrate Specificity ,Helicase ,03 medical and health sciences ,chemistry.chemical_compound ,RecQ ,Protein Domains ,Escherichia coli ,Fluorescence Resonance Energy Transfer ,Humans ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,RecQ Helicases ,030102 biochemistry & molecular biology ,biology ,E. coli ,nutritional and metabolic diseases ,Single-molecule ,DNA ,Cell Biology ,Processivity ,Recombinant Proteins ,Protein Structure, Tertiary ,Cell biology ,G-Quadruplexes ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Nucleic Acid Conformation ,Molecular Biophysics ,Protein Binding - Abstract
RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.
- Published
- 2020
29. Tuning the organelle specificity and cytotoxicity of iridium(iii) photosensitisers for enhanced phototheranostic applications
- Author
-
Zhu, Jing-Hui, primary, Xu, Guang-Xi, additional, Shum, Justin, additional, Lee, Lawrence Cho-Cheung, additional, and Lo, Kenneth Kam-Wing, additional
- Published
- 2021
- Full Text
- View/download PDF
30. Photofunctional transition metal complexes as cellular probes, bioimaging reagents and phototherapeutics
- Author
-
Xu, Guang-Xi, primary, Mak, Eunice Chiu-Lam, additional, and Lo, Kenneth Kam-Wing, additional
- Published
- 2021
- Full Text
- View/download PDF
31. Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation
- Author
-
Bo Sun, Fangzhu Wang, Xingxu Huang, Xi-Miao Hou, Fengcai Wen, Xia Zhang, Hai-Hong Li, Lulu Bi, Qian Zhang, Siqi Zhang, Lijuan Guo, Bin Shen, Xu-Guang Xi, Lille School of Management Research Center - ULR 4112 (LSMRC), SKEMA Business School-Université de Lille, Laboratory for the Modeling of Biological and Socio-technical Systems [Boston] (MoBS), Northeastern University [Boston], Department of Therapeutic Radiology, and Yale University [New Haven]
- Subjects
Staphylococcus aureus ,[SDV]Life Sciences [q-bio] ,single molecule ,Dissociative Disorders ,Chromatin, Epigenetics, Genomics & Functional Genomics ,Biochemistry ,Article ,Helicase ,03 medical and health sciences ,chemistry.chemical_compound ,Endonuclease ,0302 clinical medicine ,Bacterial Proteins ,Transcription (biology) ,Genetics ,Humans ,CRISPR ,Protein–DNA interaction ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,Gene Editing ,0303 health sciences ,biology ,Chemistry ,Cas9 ,DNA-protein interaction ,Articles ,DNA ,SaCas9 ,3. Good health ,Cell biology ,Protospacer adjacent motif ,& Genomics ,DNA‐protein interaction ,biology.protein ,single molecule Subject Categories Chromatin ,CRISPR-Cas Systems ,Transcription ,030217 neurology & neurosurgery ,RNA, Guide, Kinetoplastida - Abstract
Staphylococcus aureus Cas9 (SaCas9) is an RNA‐guided endonuclease that targets complementary DNA adjacent to a protospacer adjacent motif (PAM) for cleavage. Its small size facilitates in vivo delivery for genome editing in various organisms. Herein, using single‐molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6‐ and 18‐bp of PAM‐proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6 bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA‐bound SaCas9 a persistent block to DNA‐tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM‐distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications., This study provides a detailed understanding of SaCas9 DNA target association and dissociation and identifies two stable interactions between SaCas9 and DNA governing their interplay.
- Published
- 2020
32. Human RPA activates BLM’s bidirectional DNA unwinding from a nick
- Author
-
Xu-Guang Xi, Ming Li, Zhenheng Qin, Bo Sun, Xi-Miao Hou, Siqi Zhang, Ying Lu, Mauro Modesti, Xia Zhang, Lulu Bi, Lille School of Management Research Center - ULR 4112 (LSMRC), Université de Lille-SKEMA Business School, State Key Laboratory of Geological Processes and Mineral Resources [Wuhan] (GPMR), China University of Geosciences [Wuhan] (CUG), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Department of Therapeutic Radiology, Yale University [New Haven], SKEMA Business School-Université de Lille, Aix Marseille Université (AMU)-Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
DNA repair ,QH301-705.5 ,Structural Biology and Molecular Biophysics ,chicken ,Science ,[SDV]Life Sciences [q-bio] ,DNA, Single-Stranded ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,single molecule ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Replication Protein A ,molecular biophysics ,structural biology ,DNA unwinding ,DNA Breaks, Single-Stranded ,Biology (General) ,Replication protein A ,Gene ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,RecQ Helicases ,General Immunology and Microbiology ,biology ,optical tweezers ,General Neuroscience ,030302 biochemistry & molecular biology ,DNA replication ,Helicase ,General Medicine ,Cell biology ,helicase ,DNA Topoisomerases, Type I ,Structural biology ,chemistry ,biology.protein ,Medicine ,DNA ,BLM ,RPA ,Research Article - Abstract
International audience; BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.
- Published
- 2020
33. Author response: Human RPA activates BLM’s bidirectional DNA unwinding from a nick
- Author
-
Zhenheng Qin, Mauro Modesti, Siqi Zhang, Ying Lu, Xu-Guang Xi, Xia Zhang, Lulu Bi, Ming Li, Bo Sun, and Xi-Miao Hou
- Subjects
Chemistry ,DNA unwinding ,Cell biology - Published
- 2020
34. The N-terminal of NBPF15 causes multiple types of aggregates and mediates phase transition
- Author
-
Xue-Xue Guo, Han Wu, Stéphane Réty, Xu-Guang Xi, Liu-Tao Zhai, Ecole des Hautes Etudes Commerciales (HEC Paris), Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
- Subjects
Repetitive Sequences, Amino Acid ,Circular dichroism ,[SDV]Life Sciences [q-bio] ,Gene Expression ,Biochemistry ,Phase Transition ,Protein Aggregates ,03 medical and health sciences ,0302 clinical medicine ,Microscopy, Electron, Transmission ,Protein Domains ,X-Ray Diffraction ,Dynamic light scattering ,Scattering, Small Angle ,Humans ,Gene family ,Cognitive Dysfunction ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,Coiled coil ,0303 health sciences ,Chemistry ,Small-angle X-ray scattering ,Circular Dichroism ,Breakpoint ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,N-terminus ,Biophysics ,030217 neurology & neurosurgery ,Function (biology) - Abstract
International audience; The neuroblastoma breakpoint family (NBPF) consists of 24 members that play an important role in neuroblastoma and other cancers. NBPF is an evolutionarily recent gene family that encodes several repeats of Olduvai domain and an abundant N-terminal region. The function and biochemical properties of both Olduvai domain and the N-terminal region remain enigmatic. Human NBPF15 encodes a 670 AA protein consisting of six clades of Olduvai domains. In this study, we synthesized and expressed full-length NBPF15, and purified a range of NBPF15 truncations which were analyzed using dynamic light scattering (DLS), superdex200 (S200), small-angle X-ray scattering (SAXS), far-UV circular dichroism (CD) spectroscopy, transmission electron microscope (TEM), and crystallography. We found that proteins containing both the N-terminal region and Olduvai domain are heterogeneous with multiple types of aggregates, and some of them underwent a liquid-to-solid phase transition, probably because of the entanglement within the N-terminal coiled-coil. Proteins that contain only the Olduvai domain are homogeneous extended monomers, and those with the conserved clade 1 (CON1) have manifested a tendency to crystallize. We suggest that the entanglements between the mosaic disorder-ordered segments in NBPF15 N terminus have triggered the multiple types of aggregates and phase transition of NBPF15 proteins, which could be associated with Olduvai-related cognitive dysfunction diseases.
- Published
- 2020
35. DDX43 prefers single strand substrate and its full binding activity requires physical connection of all domains
- Author
-
Peng-Yang Chen, Liu-Tao Zhai, Han Wu, Xu-Guang Xi, Rennes School of Business, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), and École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,DEAD box ,[SDV]Life Sciences [q-bio] ,Biophysics ,DNA, Single-Stranded ,Guanosine ,In Vitro Techniques ,Biochemistry ,Substrate Specificity ,DEAD-box RNA Helicases ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Catalytic Domain ,Humans ,Protein Interaction Domains and Motifs ,Nucleotide ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,Binding Sites ,biology ,Helicase ,RNA ,Cell Biology ,RNA Helicase A ,Recombinant Proteins ,KH domain ,Neoplasm Proteins ,3. Good health ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,DNA - Abstract
International audience; DDX43 is a cancer/testis antigen and is thought to stimulate oncogenic pathways in cell proliferation, while its specific function in cancer development is largely unexplored. DDX43 is the member of RNA helicase in DEAD-box family, consists of conserved helicase core and a single K-homology (KH) domain in its N-terminus. In this paper, we expressed and purified human DDX43 protein in E. coli and demonstrated that this protein is a homogeneous monomer. To understand the role and explore the substrates preference of DDX43 in vitro, we systematically studied its binding properties. We found that DDX43 prefers single-strand DNA or RNA with length longer than 12 nt and much prefers guanosine than the other three nucleotides. Achievement of the full binding affinity of protein to substrate needs the existence of all domains, and they must be connected. The absence of either of them or the disjunction can result in a decreased binding affinity to substrates, approximately reduced 10-fold. We also found that the unwinding ability of DDX43 in vitro was neither efficient nor sustainable.
- Published
- 2019
36. The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation
- Author
-
Bo Sun, Xingxu Huang, Qian Zhang, Jiachuan Jin, Xu-Guang Xi, Bin Shen, Siqi Zhang, Ying Lu, Lulu Bi, Fengcai Wen, Ming Li, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology., State Key Laboratory of Chemical Resource Engineering, Kamerlingh Onnes Laboratorium (KOL), LION-Rijksuniversiteit Leiden, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), ShanghaiTech University [Shanghai], Shanghai Institute of Biochemistry and Cell Biology [Shanghai, China], School of Life Science and Technology [Sichuan], Southwest University of Science and Technology [Mianyang] (SWUST), Nanjing Medical University, and University of Chinese Academy of Sciences [Beijing] (UCAS)
- Subjects
Base pair ,[SDV]Life Sciences [q-bio] ,Biophysics ,02 engineering and technology ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,Endonuclease ,CRISPR-Associated Protein 9 ,CRISPR ,Nucleotide Motifs ,Molecular Biology ,Research Articles ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Multidisciplinary ,Models, Genetic ,biology ,Cas9 ,SciAdv r-articles ,Life Sciences ,Helicase ,DNA ,Sequence Analysis, DNA ,021001 nanoscience & nanotechnology ,Protospacer adjacent motif ,chemistry ,biology.protein ,Nucleic Acid Conformation ,CRISPR-Cas Systems ,0210 nano-technology ,Research Article ,Protein Binding ,RNA, Guide, Kinetoplastida - Abstract
This work determines the interactions between spCas9/sgRNA and DNA that dictate spCas9 binding and dissociation., Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.
- Published
- 2019
37. Insights into the structural and mechanistic basis of multifunctional S. cerevisiae Pif1p helicase
- Author
-
Shuo-Xing Dou, Wen-Qiang Wu, Xu-Guang Xi, Wei-Fei Chen, Dan Li, Hai-Yun Ma, Na-Nv Liu, Ke-Yu Lu, Yang-Xue Dai, Stéphane Réty, Northwest A and F University, Laboratoire de biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Chinese Academy of Sciences [Beijing] (UCAS), Beijing National Laboratory for Condensed Matter Physics and Institute of Physics (IoP/CAS), Chinese Academy of Sciences [Changchun Branch] (CAS), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), BASICS, Shanghai Jiao Tong University [Shanghai], Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, xi, Xu-Guang, École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Cachan (ENS Cachan)
- Subjects
Protein Conformation, alpha-Helical ,0301 basic medicine ,Genome instability ,[SDV]Life Sciences [q-bio] ,Gene Expression ,Crystallography, X-Ray ,Substrate Specificity ,chemistry.chemical_compound ,Adenosine Triphosphate ,Structural Biology ,Cloning, Molecular ,DNA, Fungal ,[SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,ComputingMilieux_MISCELLANEOUS ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Hydrolysis ,Recombinant Proteins ,Molecular Docking Simulation ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Biochemistry ,Thermodynamics ,Protein Binding ,Saccharomyces cerevisiae Proteins ,[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Saccharomyces cerevisiae ,DNA, Single-Stranded ,Molecular Dynamics Simulation ,Biology ,03 medical and health sciences ,Hydrolase ,Escherichia coli ,Genetics ,Protein Interaction Domains and Motifs ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Binding site ,Binding Sites ,Sequence Homology, Amino Acid ,DNA Helicases ,Helicase ,Ribosomal RNA ,biology.organism_classification ,G-Quadruplexes ,DNA binding site ,Kinetics ,030104 developmental biology ,chemistry ,biology.protein ,Protein Conformation, beta-Strand ,Protein Multimerization ,Sequence Alignment ,DNA - Abstract
International audience; The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237−780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.
- Published
- 2017
38. Photo‐ and Electrochemical Dual‐Responsive Iridium Probe for Saccharide Detection.
- Author
-
Carrod, Andrew J., Graglia, Francesco, Male, Louise, Le Duff, Cécile, Simpson, Peter, Elsherif, Mohamed, Ahmed, Zubair, Butt, Haider, Xu, Guang‐Xi, Kam‐Wing Lo, Kenneth, Bertoncello, Paolo, and Pikramenou, Zoe
- Subjects
SACCHARIDES ,IRIDIUM ,MONOSACCHARIDES ,BORONIC acids ,AQUEOUS solutions ,LUMINESCENCE ,CANCER cells - Abstract
Dual detection systems are of interest for rapid, accurate data collection in sensing systems and in vitro testing. We introduce an IrIII complex with a boronic acid receptor site attached to the 2‐phenylpyridine ligand as an ideal probe with photo‐ and electrochemical signals that is sensitive to monosaccharide binding in aqueous solution. The complex displays orange luminescence at 618 nm, which is reduced by 70 and 40 % upon binding of fructose and glucose, respectively. The electro‐chemiluminescent signal of the complex also shows a direct response to monosaccharide binding. The IrIII complex shows the same response upon incorporation into hydrogel matrices as in solution, thus demonstrating the potential of its integration into a device, as a nontoxic, simple‐to‐use tool to observe sugar binding over physiologically relevant pH ranges and saccharide concentrations. Moreover, the complex's luminescence is responsive to monosaccharide presence in cancer cells. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
39. Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA
- Author
-
Bo Sun, Ting-Ting Guo, Ya-Ting Zheng, Yi-Ran Wang, Chang-Wei Lai, Xi-Miao Hou, Xu-Guang Xi, Northwest A and F University, ShanghaiTech University [Shanghai], Laboratoire de biologie et pharmacologie appliquée (LBPA), and Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay)
- Subjects
0301 basic medicine ,Genome instability ,Molecular biology ,Science ,02 engineering and technology ,G-quadruplex ,complex mixtures ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Replication fork stalling ,Replication protein A ,Multidisciplinary ,biology ,Helicase ,021001 nanoscience & nanotechnology ,3. Good health ,Cell biology ,Folding (chemistry) ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Förster resonance energy transfer ,chemistry ,biology.protein ,0210 nano-technology ,Molecular structure ,DNA - Abstract
Summary G-quadruplexes (G4s) are non-canonical DNA structures with critical roles in DNA metabolisms. To resolve those structures that can cause replication fork stalling and genomic instability, single-stranded DNA-binding proteins and helicases are required. Here, we characterized the interplay between RPA and helicases on G4s using single-molecule FRET. We first discovered that human RPA efficiently prevents G4 formation by preempting ssDNA before its folding. RPA also differentially interacts with the folded G4s. However, helicases such as human BLM and yeast Pif1 have different G4 preferences from RPA mainly based on loop lengths. More importantly, both RPA and these helicases are required for the stable G4 unfolding, as RPA promotes helicase-mediated repetitive unfolding into durative linear state. Furthermore, BLM can traverse G4 obstacles temporarily disrupted by RPA and continue to unwind downstream duplex. We finally proposed the mechanisms underlying above functions of RPA in preventing, resolving, and assisting helicases to eliminate G4s., Graphical abstract, Highlights • RPA efficiently prevents G4 formation by preempting ssDNA before its folding • Loop length may direct folded G4s to different unfolding way by RPA and helicases • RPA promotes helicase-mediated repetitive G4 unfolding into durative linear state • RPA assists BLM to overcome G4 obstacle and continue to unwind downstream duplex, Molecular structure; Molecular biology
- Published
- 2021
40. AvrXa27 binding influences unwinding of the double-stranded DNA in the UPT box
- Author
-
Jing Zhao, Xu-Guang Xi, Jing Fu, Junpeng Jiang, Qi Wei, Na-Nv Liu, and Bo Zhang
- Subjects
0106 biological sciences ,0301 basic medicine ,Xanthomonas ,Biophysics ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,TAL effector ,Transcription (biology) ,Molecular Biology ,Genetics ,Transcription activator-like effector nuclease ,Base Sequence ,biology ,urogenital system ,Effector ,Wild type ,Helicase ,DNA ,Cell Biology ,biology.organism_classification ,Kinetics ,030104 developmental biology ,chemistry ,biology.protein ,Nucleic Acid Conformation ,Protein Binding ,010606 plant biology & botany - Abstract
Transcription-Activator Like (TAL) effectors, delivered by Xanthomonas pathogens bind specifically to UP-regulated by TAL effectors (UPT) box of the host gene promoter to arouse disease or trigger defense response. This type of protein-DNA interaction model has been applied in site-directed genome editing. However, the off-target effects of TAL have severely hindered the development of this promising technology. To better exploit the specific interaction and to deeper understand the TAL-induced host transcription rewiring, the binding between the central repeat region (CRR) of the TAL effector AvrXa27 and its UPT box variants was studied by kinetics analysis and TAL-blocked helicase unwinding assay. The results revealed that while AvrXa27 exhibited the highest affinity to the wild type UPT box, it could also bind to mutated UPT box variants, implying the possibility of non-specific interactions. Furthermore, some of these non-specific combinations restricted the helicase-elicited double-stranded DNA (dsDNA) separation to a greater extent. Our findings provide insight into the mechanism of TAL transcriptional activation and are beneficial to TAL-mediated genome modification.
- Published
- 2017
41. Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures
- Author
-
Xu-Guang Xi, Yi-Ran Wang, Bo Gao, Qing-Man Wang, Yantao Yang, Xi-Miao Hou, and École normale supérieure - Cachan (ENS Cachan)
- Subjects
0301 basic medicine ,[SDV]Life Sciences [q-bio] ,genetic processes ,DNA, Single-Stranded ,Fluorescence Polarization ,DNA and Chromosomes ,environment and public health ,complex mixtures ,Biochemistry ,DNA-binding protein ,03 medical and health sciences ,chemistry.chemical_compound ,Replication Protein A ,Fluorescence Resonance Energy Transfer ,Humans ,Protein–DNA interaction ,Molecular Biology ,Replication protein A ,ComputingMilieux_MISCELLANEOUS ,030102 biochemistry & molecular biology ,Chemistry ,DNA replication ,RNA ,Cell Biology ,Recombinant Proteins ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Förster resonance energy transfer ,Chromatography, Gel ,health occupations ,Biophysics ,Fluorescence anisotropy ,DNA ,Protein Binding - Abstract
International audience; Edited by Patrick Sung Replication protein A (RPA) is the major eukaryotic ssDNAbinding protein and has essential roles in genome maintenance. RPA binds to ssDNA through multiple modes, and recent studies have suggested that the RPA-ssDNA interaction is dynamic. However, how RPA alternates between different binding modes and modifies ssDNA structures in this dynamic interaction remains unknown. Here, we used single-molecule FRET to systematically investigate the interaction between human RPA and ssDNA. We show that RPA can adopt different types of binding complexes with ssDNAs of different lengths, leading to the straightening or bending of the ssDNAs, depending on both the length and structure of the ssDNA substrate and the RPA concentration. Importantly, we noted that some of the complexes are highly dynamic, whereas others appear relatively static. On the basis of the above observations, we propose a model explaining how RPA dynamically engages with ssDNA. Of note, fluorescence anisotropy indicated that RPA can also associate with RNA but with a lower binding affinity than with ssDNA. At the single-molecule level, we observed that RPA is undergoing rapid and repetitive associations with and dissociation from the RNA. This study may provide new insights into the rich dynamics of RPA binding to ssDNA and RNA.
- Published
- 2019
42. Construction, expression, and characterization of AG11−843 and AG11−1581
- Author
-
Xu-Guang Xi, Xie Yan, Wei Shi, Yantao Yang, Xia Ai, Institute of Zoology, Chinese Academy of Sciences [Changchun Branch] (CAS), Faculty of Business and Information Technology, University of Ontario Institute of Technology (UOIT), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), and École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Protein family ,[SDV]Life Sciences [q-bio] ,Size-exclusion chromatography ,Computational biology ,medicine.disease_cause ,lcsh:Computer applications to medicine. Medical informatics ,DNA sequencing ,law.invention ,03 medical and health sciences ,law ,medicine ,lcsh:Science (General) ,Escherichia coli ,Gene ,ComputingMilieux_MISCELLANEOUS ,Cloning ,Multidisciplinary ,Expression vector ,Chemistry ,Nucleic acid sequence ,Expression (computer science) ,DUF1220 ,030104 developmental biology ,Recombinant DNA ,lcsh:R858-859.7 ,GC-content ,Biotechnology ,lcsh:Q1-390 - Abstract
AG1, a member of the DUF1220 protein family, exhibits the most extreme human lineage-specific copy number expansion of any protein-coding sequence in the genome. These variations in copy number have been linked to both brain evolution among primates and brain size in humans. Unfortunately, our current understanding of the structure and function of these proteins is limited because current cloning and expression techniques fail to consistently produce recombinant protein for in vitro studies. The present work describes a method for amino acid and DNA sequence optimization and synthesis, recombinant protein expression and analysis of two AG1 fragments, AG11−843 and AG11−1581. It was first necessary to modify the nucleotide sequence, while holding the GC content at 52.9%. The genes were then sectionally synthesized by overlap PCR. The resulting segments were cloned into the pET-15 b-sumo expression vector and subsequently transformed into BL21 (DE3) cells. After inducing their expression, the AG11−843 and AG11−1581 proteins were isolated and purified. Furthermore, using dynamic light scattering and gel filtration analysis, AG11−843 and AG11−1581 were shown to be present in tetrameric and dimeric forms in solution. To our knowledge, this is the first study to synthesize and express fragments of the DUF1220 protein family for in vitro analysis. Taken together, the proven utility and versatility of this method indicate that it can be used as an effective technique to construct and express other proteins with complicated sequences, thus providing the means to study their function and structure in vitro.
- Published
- 2018
43. Folding Dynamics of Parallel and Antiparallel G-Triplexes under the Influence of Proximal DNA
- Author
-
Peng-Ye Wang, Jing You, Xu-Guang Xi, Hui Li, Xi-Ming Lu, Wei Li, Ming Li, Shuo-Xing Dou, China Agricultural University (CAU), Multi-disciplinary Materials Research Center, Frontier Institute of Science and Technology and State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Kamerlingh Onnes Laboratorium (KOL), LION-Rijksuniversiteit Leiden, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), and École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Circular dichroism ,Chemistry ,[SDV]Life Sciences [q-bio] ,Circular Dichroism ,DNA ,Antiparallel (biochemistry) ,7. Clean energy ,Surfaces, Coatings and Films ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Förster resonance energy transfer ,Materials Chemistry ,Biophysics ,Fluorescence Resonance Energy Transfer ,Nucleic Acid Conformation ,Physical and Theoretical Chemistry ,ComputingMilieux_MISCELLANEOUS - Abstract
The G-triplex is a kind of DNA structure formed by G-rich sequences. Previous studies have shown that it is an intermediate for the folding of G-quadruplex and has an antiparallel structure. The folding dynamics of this G-triplex structure, however, have not been well studied until now. In addition, whether a parallel G-triplex structure exists, remains unknown. In this study, by using single-molecule fluorescence resonance energy transfer and circular dichroism spectroscopy methods, we have studied the folding dynamics of the G-triplex and revealed at the single-molecule level, for the first time, that G-triplexes have both parallel and antiparallel structures. Moreover, we have investigated the effects of proximal DNA on G-triplex folding. We have found that both single-stranded TTA and double-stranded DNA at either end of a G-triplex sequence can reduce its folding speed. More interestingly, when located at the 5' end of a G-triplex sequence, the proximal DNA will favor the folding of parallel over antiparallel G-triplex structures. As G-triplex is an intermediate for G-quadruplex folding, the present results may also shed new light on the folding properties of G-quadruplex structures, in terms of dynamics, stability, and the effects of proximal DNA.
- Published
- 2018
44. Folding Kinetics of Single Human Telomeric G-Quadruplex Affected by Cisplatin
- Author
-
Xi-Miao Hou, Peng-Ye Wang, Xu-Guang Xi, Shuo-Xing Dou, Jing You, Hai-Peng Ju, Yi-Zhou Wang, Wei Li, University of Science and Technology of China [Hefei] (USTC), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, and Multi-disciplinary Materials Research Center, Frontier Institute of Science and Technology and State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University
- Subjects
0301 basic medicine ,Magnetic tweezers ,Dna duplex ,[SDV]Life Sciences [q-bio] ,General Chemical Engineering ,Kinetics ,Biology ,010402 general chemistry ,G-quadruplex ,01 natural sciences ,Article ,lcsh:Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,heterocyclic compounds ,ComputingMilieux_MISCELLANEOUS ,Cisplatin ,General Chemistry ,0104 chemical sciences ,3. Good health ,Folding (chemistry) ,030104 developmental biology ,lcsh:QD1-999 ,Biochemistry ,chemistry ,Chemotherapy Drugs ,DNA ,medicine.drug - Abstract
G-Quadruplex DNA structure has been proven to be a binding target for small molecular organic compounds and hence regarded as a promising pharmacological target. Cisplatin is a widely used chemotherapy drug that targets duplex DNA and was recently shown to react also with G-quadruplex, implying that cisplatin actually may also target G-quadruplex. In this work, we employed magnetic tweezers to investigate the influence of cisplatin on the folding kinetics of single human telomeric G-quadruplex. It was revealed that cisplatin and G-quadruplex interact in two different and competitive ways that depend on cisplatin concentration.
- Published
- 2016
45. Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi.
- Author
-
Liu-Tao Zhai, Rety, Stephane, Wei-Fei Chen, Ze-Yu Song, Auguin, Daniel, Bo Sun, Shuo-Xing Dou, and Xu-Guang Xi
- Published
- 2021
- Full Text
- View/download PDF
46. Crystal structure of Escherichia coli DEAH/RHA helicase HrpB
- Author
-
Stéphane Réty, Xu-Guang Xi, Ben-Ge Xin, Wei-Fei Chen, Yang-Xue Dai, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), and École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,[SDV]Life Sciences [q-bio] ,Amino Acid Motifs ,Biophysics ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Nucleic Acids ,Escherichia coli ,medicine ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,Binding Sites ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Nucleotides ,Helicase ,RNA ,Hydrogen Bonding ,Cell Biology ,RNA Helicase A ,Adenosine Diphosphate ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,Sequence motif ,Function (biology) ,DNA - Abstract
RNA helicases are almost ubiquitous important enzymes that take part in multiple aspects of RNA metabolism. Prokaryotes encode fewer RNA helicases than eukaryotes, suggesting that individual prokaryotic RNA helicases may take on multiple roles. The specific functions and molecular mechanisms of bacterial DEAH/RHA helicases are poorly understood, and no structures are available of these bacterial enzymes. Here, we report the first crystal structure of the DEAH/RHA helicase HrpB of Escherichia coli in a complex with ADP•AlF4. It showed an atypical globular structure, consisting of two RecA domains, an HA2 domain and an OB domain, similar to eukaryotic DEAH/RHA helicases. Notably, it showed a unique C-terminal extension that has never been reported before. Activity assays indicated that EcHrpB binds RNA but not DNA, and does not exhibit unwinding activity in vitro. Thus, within cells, the EcHrpB may function in helicase activity-independent RNA metabolic processes.
- Published
- 2018
47. Asynchrony of Base-Pair Breaking and Nucleotide Releasing of Helicases in DNA Unwinding
- Author
-
Ming Li, Chun-Hua Xu, Xu-Guang Xi, Qi Jia, Ying Lu, Dong-Fei Ma, Xing-Yuan Huang, Jing-Hua Li, Jianbing Ma, Kamerlingh Onnes Laboratorium (KOL), LION-Rijksuniversiteit Leiden, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), and École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Resonant inductive coupling ,Saccharomyces cerevisiae Proteins ,Base pair ,[SDV]Life Sciences [q-bio] ,Saccharomyces cerevisiae ,medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,0302 clinical medicine ,Escherichia coli ,Fluorescence Resonance Energy Transfer ,Materials Chemistry ,medicine ,Nucleotide ,Physical and Theoretical Chemistry ,Base Pairing ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,RecQ Helicases ,biology ,Chemistry ,DNA Helicases ,Helicase ,DNA ,biology.organism_classification ,Recombinant Proteins ,Surfaces, Coatings and Films ,Kinetics ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Förster resonance energy transfer ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,Monte Carlo Method ,030217 neurology & neurosurgery - Abstract
Helicases harness the energy of nucleotide triphosphate hydrolysis to unwind double-stranded DNA (dsDNA) in discrete steps. In spite of intensive studies, the mechanism of stepping is still poorly understood. Here, we applied single-molecule fluorescent resonant energy transfer to characterize the stepping of two nonring helicases, Escherichia coli RecQ ( E. coli RecQ) and Saccharomyces cerevisiae Pif1 (ScPif1). Our data showed that when forked dsDNA with free overhangs are used as substrates, both E. coli RecQ and ScPif1 unwind the dsDNA in nonuniform steps that distribute over broad ranges. When tension is exerted on the overhangs, the overall profile of the step-size distribution of ScPif1 is narrowed, whereas that of E. coli RecQ remains unchanged. Moreover, the measured step sizes of the both helicases concentrate on integral multiples of a half base pair. We propose a universal stepping mechanism, in which a helicase breaks one base pair at a time and sequesters the nascent nucleotides and then releases them after a random number of base-pair breaking events. The mechanism can interpret the observed unwinding patterns quantitatively and provides a general view of the helicase activity.
- Published
- 2018
48. Construction, expression, and characterization of AG1
- Author
-
Xie, Yan, Yan-Tao, Yang, Wei, Shi, Xia, Ai, and Xu-Guang, Xi
- Subjects
Base Sequence ,DNA Copy Number Variations ,AG1 ,Genetic Vectors ,Gene Expression ,Nerve Tissue Proteins ,DNA ,Sequence Analysis, DNA ,Gene synthesis ,Plasmid construction ,Recombinant protein expression ,Polymerase Chain Reaction ,Recombinant Proteins ,PCR ,Physics and Astronomy ,Protein Domains ,Chromatography, Gel ,Escherichia coli ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Protein Multimerization ,DUF1220 - Abstract
AG1, a member of the DUF1220 protein family, exhibits the most extreme human lineage-specific copy number expansion of any protein-coding sequence in the genome. These variations in copy number have been linked to both brain evolution among primates and brain size in humans. Unfortunately, our current understanding of the structure and function of these proteins is limited because current cloning and expression techniques fail to consistently produce recombinant protein for in vitro studies. The present work describes a method for amino acid and DNA sequence optimization and synthesis, recombinant protein expression and analysis of two AG1 fragments, AG1
- Published
- 2018
49. DNA-unwinding activity of Saccharomyces cerevisiae Pif1 is modulated by thermal stability, folding conformation, and loop lengths of G-quadruplex DNA
- Author
-
Xi-Miao Hou, Yi-Ran Wang, Xu-Guang Xi, Lei Wang, Qing-Man Wang, Northwest A and F University, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), and École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Hoogsteen base pair ,Saccharomyces cerevisiae ,DNA and Chromosomes ,Antiparallel (biochemistry) ,G-quadruplex ,Biochemistry ,Genomic Instability ,03 medical and health sciences ,chemistry.chemical_compound ,Fluorescence Resonance Energy Transfer ,Protein–DNA interaction ,DNA, Fungal ,Molecular Biology ,[SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology ,biology ,Chemistry ,Circular Dichroism ,DNA Helicases ,Temperature ,Helicase ,Cell Biology ,biology.organism_classification ,G-Quadruplexes ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,030104 developmental biology ,Förster resonance energy transfer ,biology.protein ,Biophysics ,DNA ,Protein Binding - Abstract
International audience; G-quadruplexes (G4s) are four-stranded DNA structures formed by Hoogsteen base pairing between stacked sets of four guanines. Pif1 helicase plays critical roles in suppressing genomic instability in the yeast Saccharomyces cerevisiae by resolving G4s. However, the structural properties of G4s in S. cerevisiae and the substrate preference of Pif1 for different G4s remain unknown. Here, using CD spectroscopy and 83 G4 motifs from S. cerevisiae ranging in length from 30 to 60 nucleotides, we first show that G4 structures can be formed with a broad range of loop sizes in vitro and that a parallel conformation is favored. Using single-molecule FRET analysis, we then systematically addressed Pif1-mediated unwinding of various G4s and found that Pif1 is sensitive to G4 stability. Moreover, Pif1 preferentially unfolded antiparallel G4s rather than parallel G4s having similar stability. Furthermore, our results indicate that most G4 structures in S. cerevisiae sequences have long loops and can be efficiently unfolded by Pif1 because of their low stability. However, we also found that G4 structures with short loops can be barely unfolded. This study highlights the formidable capability of Pif1 to resolve the majority of G4s in S. cerevisiae sequences, narrows the fractions of G4s that may be challenging for genomic stability, and provides a framework for understanding the influence of different G4s on genomic stability via their processing by Pif1.
- Published
- 2018
50. Solvothermal Synthesis and Luminescence Properties of Gd2O2S:RE3+ (RE3+=Eu3+/Tb3+) Hollow Sphere
- Author
-
Xu, Guang Xi, primary, Sang, Xiao Tong, additional, Lian, Jing Bao, additional, Wu, Nian Chu, additional, and Zhang, Xue, additional
- Published
- 2019
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.