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Biochemical Society Transactions Oct 2023Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for... (Review)
Review
Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for the generation of mature T and B cells in the adaptive immune system via the process of V(D)J recombination. Here, we review how recently determined structures shed light on how NHEJ complexes function at DNA DSBs, emphasizing how multiple structures containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) may function in NHEJ. Together, these studies provide an explanation for how NHEJ proteins assemble to detect and protect DSB ends, then proceed, through DNA-PKcs-dependent autophosphorylation, to a ligation-competent complex.
Topics: Humans; DNA-Binding Proteins; DNA End-Joining Repair; DNA Breaks, Double-Stranded; Phosphorylation; DNA; DNA Repair
PubMed: 37787023
DOI: 10.1042/BST20220741 -
Nature Communications Dec 2023Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the...
Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.
Topics: Humans; Telomeric Repeat Binding Protein 2; Telomere; Shelterin Complex; Telomere-Binding Proteins; DNA
PubMed: 38086788
DOI: 10.1038/s41467-023-43397-7 -
Trends in Biochemical Sciences Oct 2023G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can... (Review)
Review
G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can specifically bind to G4 structures. There is increasing evidence that G4-protein interactions involve in the regulation of important cellular processes, such as DNA replication, transcription, RNA splicing, and translation. Additionally, G4-protein interactions have been demonstrated to be potential targets for disease treatment. In order to unravel the detailed regulatory mechanisms of G4-binding proteins (G4BPs), biochemical methods for detecting G4-protein interactions with high specificity and sensitivity are highly demanded. Here, we review recent advances in screening and validation of new G4BPs and highlight both their features and limitations.
Topics: G-Quadruplexes; DNA; DNA Replication; RNA
PubMed: 37422364
DOI: 10.1016/j.tibs.2023.06.007 -
Nucleic Acids Research Sep 2023In meiosis, Dmc1 recombinase and the general recombinase Rad51 are responsible for pairing homologous chromosomes and exchanging strands. Fission yeast...
In meiosis, Dmc1 recombinase and the general recombinase Rad51 are responsible for pairing homologous chromosomes and exchanging strands. Fission yeast (Schizosaccharomyces pombe) Swi5-Sfr1 and Hop2-Mnd1 stimulate Dmc1-driven recombination, but the stimulation mechanism is unclear. Using single-molecule fluorescence resonance energy transfer (smFRET) and tethered particle motion (TPM) experiments, we showed that Hop2-Mnd1 and Swi5-Sfr1 individually enhance Dmc1 filament assembly on single-stranded DNA (ssDNA) and adding both proteins together allows further stimulation. FRET analysis showed that Hop2-Mnd1 enhances the binding rate of Dmc1 while Swi5-Sfr1 specifically reduces the dissociation rate during the nucleation, about 2-fold. In the presence of Hop2-Mnd1, the nucleation time of Dmc1 filaments shortens, and doubling the ss/double-stranded DNA (ss/dsDNA) junctions of DNA substrates reduces the nucleation times in half. Order of addition experiments confirmed that Hop2-Mnd1 binds on DNA to recruit and stimulate Dmc1 nucleation at the ss/dsDNA junction. Our studies directly support the molecular basis of how Hop2-Mnd1 and Swi5-Sfr1 act on different steps during the Dmc1 filament assembly. DNA binding of these accessory proteins and nucleation preferences of recombinases thus dictate how their regulation can take place.
Topics: Cell Cycle Proteins; DNA; DNA, Single-Stranded; Meiosis; Rad51 Recombinase; Recombinases; Schizosaccharomyces
PubMed: 37395447
DOI: 10.1093/nar/gkad561 -
Spectrochimica Acta. Part A, Molecular... Nov 2023Sertraline Hydrochloride (STH) is an antidepressant drug that belongs to the selective serotonin reuptake inhibitor family (SSRIs), which inhibits serotonin uptake in...
Sertraline Hydrochloride (STH) is an antidepressant drug that belongs to the selective serotonin reuptake inhibitor family (SSRIs), which inhibits serotonin uptake in presynaptic nerve fibers. The use of these medications without a legitimate prescription might result in adverse effects, and in rare circumstances, death. The interaction mechanism and binding mode of STH with duplex DNA were extensively investigated using spectroscopic and modeling techniques at different temperatures. The hypochromic shift of the absorption spectra of STH on binding with CT-DNA indicated groove binding. Fluorescence spectroscopic studies showed that CT-DNA quenches the fluorescence intensity of STH through a static quenching mechanism. The thermodynamic parameters indicated that the complex formation was spontaneous, and enthalpy driven. The competitive displacement binding study revealed that STH displaced DAPI from the minor groove of DNA. Molecular docking and molecular dynamics simulations also revealed that the complex was stable over 150 ns and that STH preferred the minor groove of DNA. The binding energy of the stable conformations were evaluated through MM/PBSA methods. A comparison of the bound poses at different timescales showed minor changes in STH structure upon DNA binding. Furthermore, a structural analysis of CT-DNA indicated that STH induced changes in the sugar-phosphate backbone had an impact on the minor groove's width which are in agreement with the CD spectroscopic results. This study provides a better understanding of STH binding with duplex DNA.
Topics: Molecular Docking Simulation; Sertraline; DNA; Spectrometry, Fluorescence; Thermodynamics; Tomography, X-Ray Computed; Circular Dichroism; Binding Sites
PubMed: 37257324
DOI: 10.1016/j.saa.2023.122910 -
Nucleic Acids Research Dec 2023Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed...
Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.
Topics: Animals; Forkhead Transcription Factors; Base Sequence; Protein Domains; Binding Sites; DNA; Mammals
PubMed: 37933840
DOI: 10.1093/nar/gkad994 -
Cell Host & Microbe Aug 2023The Gabija complex is a prokaryotic antiviral system consisting of the GajA and GajB proteins. GajA was identified as a DNA nicking endonuclease but the functions of...
The Gabija complex is a prokaryotic antiviral system consisting of the GajA and GajB proteins. GajA was identified as a DNA nicking endonuclease but the functions of GajB and the complex remain unknown. Here, we show that synergy between GajA-mediated DNA cleavage and nucleotide hydrolysis by GajB initiates efficient abortive infection defense against virulent bacteriophages. The antiviral activity of GajA requires GajB, which senses DNA termini produced by GajA to hydrolyze (d)A/(d)GTP, depleting essential nucleotides. This ATPase activity of Gabija complex is only activated upon DNA binding. GajA binds to GajB to form stable complexes in vivo and in vitro. However, a functional Gabija complex requires a molecular ratio between GajB and GajA below 1:1, indicating stoichiometric regulation of the DNA/nucleotide processing complex. Thus, the Gabija system exhibits distinct and efficient antiviral defense through sequential sensing and activation of nucleotide depletion and DNA cleavage, causing a cascade suicide effect.
Topics: Humans; Nucleotides; Antiviral Agents; DNA Cleavage; DNA; Hydrolysis
PubMed: 37480847
DOI: 10.1016/j.chom.2023.06.014 -
Journal of Biomolecular Structure &... Dec 2023Two phytochemicals, thymol and thymoquinone obtained from () and seed, respectively. Both the phytochemicals show several biochemical activities like anticancer,...
Two phytochemicals, thymol and thymoquinone obtained from () and seed, respectively. Both the phytochemicals show several biochemical activities like anticancer, antimicrobial etc. In this paper, we studied the affinities of thymol and thymoquinone towards calf thymus DNA (CT-DNA) and protein (bovine serum albumin). Spectroscopic and molecular modelling studies revealed that both compounds have a high affinity toward both the receptors; DNA and protein. Both phytochemicals binds to the minor grooves of DNA and suitable pockets of protein. Several free energy function and hydrogen bonding play significant role during the binding phenomenon.Communicated by Ramaswamy H. Sarma.
Topics: Protein Binding; Thymol; Molecular Docking Simulation; DNA; Serum Albumin, Bovine; Binding Sites; Spectrometry, Fluorescence
PubMed: 36841618
DOI: 10.1080/07391102.2023.2180665 -
Nucleic Acids Research Sep 2023The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding...
The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12-15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1-ZF7 and ZF3-ZF11 in complex with DNA that incorporates the CORE sequence together with either 3' downstream or 5' upstream motifs. Like conventional tandem ZF array proteins, ZF1-ZF7 follow the right-handed twist of the DNA, with each finger occupying and recognizing one triplet of three base pairs in the DNA major groove. ZF8 plays a unique role, acting as a spacer across the DNA minor groove and positioning ZF9-ZF11 to make cross-strand contacts with DNA. We ascribe the difference between the two subgroups of ZF1-ZF7 and ZF8-ZF11 to residues at the two positions -6 and -5 within each finger, with small residues for ZF1-ZF7 and bulkier and polar/charged residues for ZF8-ZF11. ZF8 is also uniquely rich in basic amino acids, which allows salt bridges to DNA phosphates in the minor groove. Highly specific arginine-guanine and glutamine-adenine interactions, used to recognize G:C or A:T base pairs at conventional base-interacting positions of ZFs, also apply to the cross-strand interactions adopted by ZF9-ZF11. The differences between ZF1-ZF7 and ZF8-ZF11 can be rationalized structurally and may contribute to recognition of high-affinity CTCF binding sites.
Topics: Animals; Humans; CCCTC-Binding Factor; Amino Acid Sequence; Zinc Fingers; Binding Sites; DNA; Mammals
PubMed: 37439339
DOI: 10.1093/nar/gkad594 -
Cell Chemical Biology Aug 2023CRISPR-based editing has revolutionized genome engineering despite the observation that many DNA sequences remain challenging to target. Unproductive interactions formed...
CRISPR-based editing has revolutionized genome engineering despite the observation that many DNA sequences remain challenging to target. Unproductive interactions formed between the single guide RNA's (sgRNA) Cas9-binding scaffold domain and DNA-binding antisense domain are often responsible for such limited editing resolution. To bypass this limitation, we develop a functional SELEX (systematic evolution of ligands by exponential enrichment) approach, termed BLADE (binding and ligand activated directed evolution), to identify numerous, diverse sgRNA variants that bind Streptococcus pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in sgRNA sequence. We also observe that particular variants partner more effectively with specific DNA-binding antisense domains, yielding combinations with enhanced editing efficiencies at various target sites. Using molecular evolution, CRISPR-based systems could be created to efficiently edit even challenging DNA sequences making the genome more tractable to engineering. This selection approach will be valuable for generating sgRNAs with a range of useful activities.
Topics: CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; RNA; DNA; Gene Editing
PubMed: 37390831
DOI: 10.1016/j.chembiol.2023.06.007