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Molecular Cell Feb 2024The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA...
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.
Topics: Humans; DNA; DNA Repair; DNA Replication; DNA, Cruciform; DNA-Binding Proteins; Recombinases; RecQ Helicases
PubMed: 38266639
DOI: 10.1016/j.molcel.2023.12.040 -
Nucleic Acids Research Dec 2023The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides....
The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.
Topics: Humans; DNA; DNA Mismatch Repair; DNA Repair; DNA-Binding Proteins; MutS Homolog 2 Protein; MutS Homolog 3 Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Genomic Instability
PubMed: 37930834
DOI: 10.1093/nar/gkad934 -
Methods in Molecular Biology (Clifton,... 2024Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and...
Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data.
Topics: DNA; Nanostructures; Nanotechnology; Polymers; Microscopy, Fluorescence
PubMed: 37824005
DOI: 10.1007/978-1-0716-3377-9_9 -
Biophysical Journal Aug 2023Hybridization of short nucleic acid segments (<4 nt) to single-strand templates occurs as a critical intermediate in processes such as nonenzymatic nucleic acid...
Hybridization of short nucleic acid segments (<4 nt) to single-strand templates occurs as a critical intermediate in processes such as nonenzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood because of the experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential noncanonical base-pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2-40 μs depending on the template and temperature. Dinucleotide hybridization and dehybridization involve a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.
Topics: DNA; RNA; Nucleic Acid Hybridization; Thermodynamics; Kinetics; Spectrum Analysis; Molecular Dynamics Simulation
PubMed: 37469144
DOI: 10.1016/j.bpj.2023.07.009 -
Journal of Natural Products Jul 2023Norcryptotackieine () belongs to the indoloquinoline class of alkaloids isolated from , a plant species that has been traditionally used as an antimalarial agent....
Norcryptotackieine () belongs to the indoloquinoline class of alkaloids isolated from , a plant species that has been traditionally used as an antimalarial agent. Additional structural modifications of can potentially enhance its therapeutic potency. Indoloquinolines such as cryptolepine, neocryptolepine, isocryptolepine, and neoisocryptolepine show restricted clinical applications owing to their cytotoxicity deriving from interactions with DNA. Here, we examined the effect of substitutions at the N-6 position of norcryptotackieine on the cytotoxicity, as well as structure-activity relationship studies pertaining to sequence specific DNA-binding affinities. The representative compound binds DNA in a nonintercalative/pseudointercalative fashion, in addition to nonspecific stacking on DNA, in a sequence selective manner. The DNA-binding studies clearly establish the mechanism of DNA binding by N-6-substituted norcryptotackieines and neocryptolepine. The synthesized norcryptotackieines , and known indoloquinolines were screened on different cell lines (HEK293, OVCAR3, SKOV3, B16F10, and HeLa) to assess their cytotoxicity. Norcryptotackieine (IC value of 3.1 μM) showed 2-fold less potency when compared to the natural indoloquinoline cryptolepine (IC value of 1.64 μM) in OVCAR3 (ovarian adenocarcinoma) cell lines.
Topics: Female; Humans; Alkaloids; Apoptosis; Cell Line, Tumor; DNA; HEK293 Cells; Indole Alkaloids; Ovarian Neoplasms; Quinolines; Indoles
PubMed: 37285507
DOI: 10.1021/acs.jnatprod.2c01045 -
Nature Communications Oct 2023Synthetic gene networks in mammalian cells are currently limited to either protein-based transcription factors or RNA-based regulators. Here, we demonstrate a regulatory...
Synthetic gene networks in mammalian cells are currently limited to either protein-based transcription factors or RNA-based regulators. Here, we demonstrate a regulatory approach based on circular single-stranded DNA (Css DNA), which can be used as an efficient expression vector with switchable activity, enabling gene regulation in mammalian cells. The Css DNA is transformed into its double-stranded form via DNA replication and used as vectors encoding a variety of different proteins in a wide range of cell lines as well as in mice. The rich repository of DNA nanotechnology allows to use sort single-stranded DNA effectors to fold Css DNA into DNA nanostructures of different complexity, leading the gene expression to programmable inhibition and subsequently re-activation via toehold-mediated strand displacement. The regulatory strategy from Css DNA can thus expand the molecular toolbox for the realization of synthetic regulatory networks with potential applications in genetic diagnosis and gene therapy.
Topics: Animals; Mice; DNA, Single-Stranded; DNA; DNA Replication; DNA-Binding Proteins; Gene Expression; Mammals
PubMed: 37863879
DOI: 10.1038/s41467-023-42437-6 -
Nucleic Acids Research Aug 2023The minichromosomal maintenance proteins, MCM8 and MCM9, are more recent evolutionary additions to the MCM family, only cooccurring in selected higher eukaryotes....
The minichromosomal maintenance proteins, MCM8 and MCM9, are more recent evolutionary additions to the MCM family, only cooccurring in selected higher eukaryotes. Mutations in these genes are directly linked to ovarian insufficiency, infertility, and several cancers. MCM8/9 appears to have ancillary roles in fork progression and recombination of broken replication forks. However, the biochemical activity, specificities and structures have not been adequately illustrated, making mechanistic determination difficult. Here, we show that human MCM8/9 (HsMCM8/9) is an ATP dependent DNA helicase that unwinds fork DNA substrates with a 3'-5' polarity. High affinity ssDNA binding occurs in the presence of nucleoside triphosphates, while ATP hydrolysis weakens the interaction with DNA. The cryo-EM structure of the HsMCM8/9 heterohexamer was solved at 4.3 Å revealing a trimer of heterodimer configuration with two types of interfacial AAA+ nucleotide binding sites that become more organized upon binding ADP. Local refinements of the N or C-terminal domains (NTD or CTD) improved the resolution to 3.9 or 4.1 Å, respectively, and shows a large displacement in the CTD. Changes in AAA+ CTD upon nucleotide binding and a large swing between the NTD and CTD likely implies that MCM8/9 utilizes a sequential subunit translocation mechanism for DNA unwinding.
Topics: Humans; DNA; DNA Replication; DNA-Binding Proteins; Eukaryota; Nucleotides; DNA Helicases; Cryoelectron Microscopy
PubMed: 37309874
DOI: 10.1093/nar/gkad508 -
Angewandte Chemie (International Ed. in... Sep 2023Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed...
Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction can be directed to build Cu-binding AMN scaffolds. We selected biologically inert reaction partners tris(azidomethyl)mesitylene and ethynyl-thiophene to develop TC-Thio, a bioactive C -symmetric ligand in which three thiophene-triazole moieties are positioned around a central mesitylene core. The ligand was characterised by X-ray crystallography and forms multinuclear Cu and Cu complexes identified by mass spectrometry and rationalised by density functional theory (DFT). Upon Cu coordination, Cu -TC-Thio becomes a potent DNA binding and cleaving agent. Mechanistic studies reveal DNA recognition occurs exclusively at the minor groove with subsequent oxidative damage promoted through a superoxide- and peroxide-dependent pathway. Single molecule imaging of DNA isolated from peripheral blood mononuclear cells shows that the complex has comparable activity to the clinical drug temozolomide, causing DNA damage that is recognised by a combination of base excision repair (BER) enzymes.
Topics: Click Chemistry; Copper; Leukocytes, Mononuclear; Ligands; DNA; Azides
PubMed: 37338105
DOI: 10.1002/anie.202305759 -
Journal of the American Chemical Society Feb 2024Two-dimensional (2D) DNA origami assembly represents a powerful approach to the programmable design and construction of advanced 2D materials. Within the context of...
Two-dimensional (2D) DNA origami assembly represents a powerful approach to the programmable design and construction of advanced 2D materials. Within the context of hybridization-mediated 2D DNA origami assembly, DNA spacers play a pivotal role as essential connectors between sticky-end regions and DNA origami units. Here, we demonstrated that programming the spacer length, which determines the binding radius of DNA origami units, could effectively tune sticky-end hybridization reactions to produce distinct 2D DNA origami arrays. Using DNA-PAINT super-resolution imaging, we unveiled the significant impact of spacer length on the hybridization efficiency of sticky ends for assembling square DNA origami (SDO) units. We also found that the assembly efficiency and pattern diversity of 2D DNA origami assemblies were critically dependent on the spacer length. Remarkably, we realized a near-unity yield of ∼98% for the assembly of SDO trimers and tetramers via this spacer-programmed strategy. At last, we revealed that spacer lengths and thermodynamic fluctuations of SDO are positively correlated, using molecular dynamics simulations. Our study thus paves the way for the precision assembly of DNA nanostructures toward higher complexity.
Topics: DNA, Intergenic; Nucleic Acid Conformation; DNA; Nanostructures; Nucleic Acid Hybridization; Nanotechnology
PubMed: 38355136
DOI: 10.1021/jacs.3c13180 -
Current Opinion in Structural Biology Feb 2024Eukaryotic transcription factors activate gene expression with their DNA-binding domains and activation domains. DNA-binding domains bind the genome by recognizing... (Review)
Review
Eukaryotic transcription factors activate gene expression with their DNA-binding domains and activation domains. DNA-binding domains bind the genome by recognizing structurally related DNA sequences; they are structured, conserved, and predictable from protein sequences. Activation domains recruit chromatin modifiers, coactivator complexes, or basal transcriptional machinery via structurally diverse protein-protein interactions. Activation domains and DNA-binding domains have been called independent, modular units, but there are many departures from modularity, including interactions between these regions and overlap in function. Compared to DNA-binding domains, activation domains are poorly understood because they are poorly conserved, intrinsically disordered, and difficult to predict from protein sequences. This review, organized around commonly asked questions, describes recent progress that the field has made in understanding the sequence features that control activation domains and predicting them from sequence.
Topics: Transcriptional Activation; Protein Binding; Transcription Factors; Protein Domains; DNA
PubMed: 38056064
DOI: 10.1016/j.sbi.2023.102732