-
Acta Crystallographica. Section F,... May 2022The CENP-SX (MHF) complex is a conserved histone-fold protein complex that is involved in chromosome segregation and DNA repair. It can bind to DNA on its own as well as...
The CENP-SX (MHF) complex is a conserved histone-fold protein complex that is involved in chromosome segregation and DNA repair. It can bind to DNA on its own as well as in complex with other proteins such as CENP-TW and FANCM to recognize specific substrates. CENP-SX binds nonspecifically to dsDNA, similar to other histone-fold proteins. Several low-resolution structures of CENP-SX in complex with DNA are known, but a high-resolution structure is still lacking. The DNA-binding properties of CENP-SX and FANCM-CENP-SX complexes with various lengths of dsDNA were compared and the band-shift patterns and migration positions were found to differ. To confirm the DNA-binding properties in detail, CENP-SX-DNA and FANCM-CENP-SX-DNA complexes were crystallized. Analysis of the crystals revealed that they all contained the CENP-SX-DNA complex, irrespective of the complex that was used in crystallization. Detailed diffraction data analyses revealed that there were two types of crystal with different space groups, P2 and C2, where the volume of the P2 asymmetric unit is twice as large as that of the C2 asymmetric unit. Analysis of the self-rotation function revealed the presence of twofold and fourfold symmetry in both crystals. This suggests that there may be multiple molecules of CENP-SX and DNA within the asymmetric unit with respective symmetry. Structure determination of the present crystals should reveal details of the DNA-binding properties of CENP-SX.
Topics: Crystallization; Crystallography, X-Ray; DNA; DNA-Binding Proteins; Histones
PubMed: 35506764
DOI: 10.1107/S2053230X22003843 -
Molecular Cell Jan 2023Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer....
Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer. SPRTN is activated after the replicative CMG helicase bypasses a DPC and polymerase extends the nascent strand to the adduct. Here, we identify a role for the 5'-to-3' helicase FANCJ in DPC repair. In addition to supporting CMG bypass, FANCJ is essential for SPRTN activation. FANCJ binds ssDNA downstream of the DPC and uses its ATPase activity to unfold the protein adduct, which exposes the underlying DNA and enables cleavage of the adduct. FANCJ-dependent DPC unfolding is also essential for translesion DNA synthesis past DPCs that cannot be degraded. In summary, our results show that helicase-mediated protein unfolding enables multiple events in DPC repair.
Topics: DNA; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; DNA-Binding Proteins; Protein Unfolding
PubMed: 36608669
DOI: 10.1016/j.molcel.2022.12.005 -
Acta Crystallographica. Section D,... Apr 2010Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and...
Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
Topics: Crystallography, X-Ray; DNA; Models, Molecular; Nucleic Acid Conformation; Protein Structure, Tertiary; Proteins; RNA; Software Design
PubMed: 20383002
DOI: 10.1107/S0907444910007493 -
Biosensors Feb 2023Eltrombopag is a powerful adjuvant anticancer drug used in treating MS (myelodysplastic syndrome) and AML (acute myeloid leukemia) diseases. In this study, the...
Eltrombopag is a powerful adjuvant anticancer drug used in treating MS (myelodysplastic syndrome) and AML (acute myeloid leukemia) diseases. In this study, the interaction mechanism between eltrombopag and DNA was studied by voltammetry, spectroscopic techniques, and viscosity measurements. We developed a DNA-based biosensor and nano-biosensor using reduced graphene oxide-modified glassy carbon electrode to detect DNA-eltrombopag binding. The reduction of desoxyguanosine (dGuo) and desoxyadenosine (dAdo) oxidation signals in the presence of the drug demonstrated that a strong interaction could be established between the eltrombopag and dsDNA. The eltrombopag-DNA interaction was further investigated by UV absorption and fluorescence emission spectroscopy to gain more quantitative insight on binding. Viscosity measurements were utilized to characterize the binding mode of the drug. To shed light on the noncovalent interactions and binding mechanism of eltrombopag molecular docking and molecular dynamics (MD), simulations were performed. Through simultaneously carried out experimental and in silico studies, it was established that the eltrombopag binds onto the DNA via intercalation.
Topics: Molecular Docking Simulation; DNA; Antineoplastic Agents; Spectrometry, Fluorescence; Biosensing Techniques; Electrochemical Techniques
PubMed: 36979512
DOI: 10.3390/bios13030300 -
Molecules (Basel, Switzerland) Jul 2015While the structure of the DNA-binding domain (DBD) of the vitamin D receptor (VDR) has been determined in great detail, the roles of its domains and how to bind the... (Review)
Review
While the structure of the DNA-binding domain (DBD) of the vitamin D receptor (VDR) has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE), at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR), while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE). For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers-VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110) of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH)2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH)2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH)2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.
Topics: Amino Acid Sequence; Animals; DNA; DNA-Binding Proteins; Humans; Models, Molecular; Protein Structure, Tertiary; Receptors, Calcitriol; Structure-Activity Relationship; Zinc Fingers
PubMed: 26198224
DOI: 10.3390/molecules200712389 -
Nature Nov 2020Nucleic acids derived from pathogens induce potent innate immune responses. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor that catalyses the synthesis...
Nucleic acids derived from pathogens induce potent innate immune responses. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor that catalyses the synthesis of the cyclic dinucleotide cyclic GMP-AMP, which mediates the induction of type I interferons through the STING-TBK1-IRF3 signalling axis. cGAS was previously thought to not react with self DNA owing to its cytosolic localization; however, recent studies have shown that cGAS is localized mostly in the nucleus and has low activity as a result of tight nuclear tethering. Here we show that cGAS binds to nucleosomes with nanomolar affinity and that nucleosome binding potently inhibits its catalytic activity. To elucidate the molecular basis of cGAS inactivation by nuclear tethering, we determined the structure of mouse cGAS bound to human nucleosome by cryo-electron microscopy. The structure shows that cGAS binds to a negatively charged acidic patch formed by histones H2A and H2B via its second DNA-binding site. High-affinity nucleosome binding blocks double-stranded DNA binding and maintains cGAS in an inactive conformation. Mutations of cGAS that disrupt nucleosome binding alter cGAS-mediated signalling in cells.
Topics: Animals; Biocatalysis; Catalytic Domain; Cell Line; Cryoelectron Microscopy; DNA; Humans; Mice; Models, Molecular; Mutation; Nucleosomes; Nucleotidyltransferases; Protein Binding; Signal Transduction
PubMed: 32911481
DOI: 10.1038/s41586-020-2749-z -
International Journal of Biological... Sep 2022The lysine (K) tRNA synthetase C-terminal (KTSC) domain containing proteins are widely spread in Bacteria, Archaea and Viruses, but the function of this short domain is...
The lysine (K) tRNA synthetase C-terminal (KTSC) domain containing proteins are widely spread in Bacteria, Archaea and Viruses, but the function of this short domain is unclear. The occurrence of the fusion of KTSC domain to a catalytic domain or domains related to DNA or RNA metabolisms suggests its potential role in DNA or RNA binding. Here, we report the characterization of Mvu8s from Methanolobus vulcani, which consists of a single KTSC domain. Mvu8s binds specifically to ssDNA with an affinity approximately 40- and 10-fold higher than those for dsDNA and ssRNA in vitro, respectively. It shows a slight preference to the G-rich DNA sequence but barely binds the A-stretch. Crystal structure of Mvu8s shows that it forms a homo-tetramer, with each monomer composed of a four-strand antiparallel β-sheet and a helix-turn-helix in the order of β1-β2-β3-α1-α2-β4. Four basic residues (R3, R7, K54 and K58) were found to serve important roles in ssDNA-binding. And, the spiral arrangement of the DNA interfaces in Mvu8s homo-tetramer presumably results in ssDNA wrapping. Our results not only offer clues of the functions of the KTSC domain containing proteins but also expand our knowledge on the non-oligonucleotide-binding (OB) fold single-stranded DNA-binding proteins in Archaea.
Topics: Catalytic Domain; DNA; DNA, Single-Stranded; DNA-Binding Proteins; Protein Binding; RNA
PubMed: 35809674
DOI: 10.1016/j.ijbiomac.2022.07.015 -
International Journal of Molecular... Nov 2019The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with... (Review)
Review
The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 5'RRRCWWGYYY3' sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions.
Topics: Amino Acid Sequence; Animals; Binding Sites; Consensus Sequence; DNA; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Tumor Suppressor Protein p53
PubMed: 31717504
DOI: 10.3390/ijms20225605 -
Nucleic Acids Research Jul 2021The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each...
The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.
Topics: Binding Sites; Cells, Cultured; Cytosine; DNA; DNA Methylation; Mutation; Nucleotide Motifs; PAX7 Transcription Factor; Protein Binding; Protein Domains; Transcriptional Activation
PubMed: 34197620
DOI: 10.1093/nar/gkab561 -
International Journal of Molecular... Feb 2017p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction... (Review)
Review
p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells.
Topics: Animals; Binding Sites; DNA; DNA, B-Form; Humans; Nucleic Acid Conformation; Protein Binding; Structure-Activity Relationship; Tumor Suppressor Protein p53
PubMed: 28208646
DOI: 10.3390/ijms18020375