The active DNA-PK holoenzyme occupies a tensed state in a staggered synaptic complex.
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ABSTRACT:
SUBMITTER: Hepburn M
PROVIDER: S-EPMC8675206 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical role in non-homologous end joining (NHEJ), the major DNA repair pathway. Here, we determined cryo-electron microscopy structure of human DNA-PK holoenzyme at 6.6 Å resolution. In the complex structure, DNA-PKcs, KU70, KU80 and DNA duplex form a 650-kDa heterotetramer with 1:1:1:1 stoichiometry. The N-terminal ?-solenoid (?2 800 residues) of DNA-PKcs adopts a double-ring fold and connects the catalytic core domain of DNA-PKcs and KU70/80-DNA. DNA-PKcs and KU70/80 together form a DNA-binding tunnel, which cradles ?30-bp DNA and prevents sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing. Structural and biochemical analyses indicate that KU70/80 and DNA coordinately induce conformational changes of DNA-PKcs and allosterically stimulate its kinase activity. We propose a model for activation of DNA-PKcs in which allosteric signals are generated upon DNA-PK holoenzyme formation and transmitted to the kinase domain through N-terminal HEAT repeats and FAT domain of DNA-PKcs. Our studies suggest a mechanism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activation of DNA-PKcs and DNA-PK-mediated NHEJ pathway.
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Project description:DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4.4-Å resolution and the DNA-PK holoenzyme at 5.8-Å resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge, the circular cradle, and the head that includes the kinase domain. Two perpendicular apertures exist in the structure, which are sufficiently large for the passage of dsDNA. The DNA-PK holoenzyme cryo-EM map reveals density for the C-terminal globular domain of Ku80 that interacts with the arm of DNA-PKcs. The Ku80-binding site is adjacent to the previously identified density for the DNA-binding region of the Ku70/Ku80 complex, suggesting concerted DNA interaction by DNA-PKcs and the Ku complex.
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Project description:The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions.
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Project description:The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery. Pseudomonas aeruginosa is one of the leading causes of hospital and community acquired infections worldwide - making the organism an important public health pathogen. We developed a method for producing high quantities of highly pure and active recombinant P. aeruginosa str. PAO1 RNAP core and holoenzyme complexes that employed two-vector systems for expressing the core enzyme (?, ?, ?', and ? subunits) and for expressing the holoenzyme complex (core + ?70). Unlike other RNAP expression approaches, we used a low temperature autoinduction system in E. coli with T7 promoters that produced high cell yields and stable protein expression. The purification strategy comprised of four chromatographic separation steps (metal chelate, heparin, and ion-exchange) with yields of up to 11?mg per 500?mL culture. Purified holoenzyme and reconstituted holoenzyme from core and ?70 were highly active at transcribing both small and large-sized DNA templates, with a determined elongation rate of ~18?nt/s for the holoenzyme. The successful purification of the P. aeruginosa RNAP provides a gateway for studies focusing on in vitro transcriptional regulation in this pathogen.
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Project description:The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA-PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.
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Project description:Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.
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Project description:Ionizing radiation produces a distinctive pattern of bistranded clustered lesions in DNA. A relatively low number of clustered lesions may be lethal to cells when compared to a larger number of single lesions. Enzyme cleavage experiments suggest that the orientation of bistranded lesions causes differential recognition and removal of these lesions. Like that of a previous study of bistranded abasic site lesion [Hazel, R. D., Tian, K., and de los Santos, C. (2008) Biochemistry 47, 11909-11919], the aim of this investigation was to determine the structures of two DNA duplexes each containing two synthetic apurinic/apyrimidinic (AP) residues, positioned on opposite strands and separated by two base pairs. In the first duplex, the AP residues are staggered in the 3' orientation [-3 duplex, (AP)(2)-3 duplex], while in the second duplex, the AP residues are staggered in the 5' orientation [+3 duplex, (AP)(2)+3 duplex]. NOESY spectra recorded in 100 and 10% D(2)O buffer solutions allowed the assignment of the nonexchangeable and exchangeable protons, respectively, for each duplex. Cross-peak connectivity in the nonexchangeable proton spectra indicates that the duplex is a regular right-handed helix with the AP residues and orphan bases located inside the duplexes. The exchangeable proton spectra establish the formation of Watson-Crick G·C alignment for the two base pairs between the lesion sites in both duplexes. Distance-restrained molecular dynamics simulation confirmed the intrahelical orientations of the AP residues. The proximity of the AP residues across the minor groove of the -3 duplex and across the major groove in the +3 duplex is similar to their locations in the case of -1 and +1 clusters. This difference in structure may be a key factor in the differential recognition of bistranded AP lesions by human AP endonuclease.
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Project description:The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100-base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C-containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes.
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Project description:RATIONALE:Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding. OBJECTIVES:The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats. METHODS:Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer (11)C-SA4503 (sigma-1R) or (11)C-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with (11)C-raclopride. Cerebral (11)C-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V T) calculated by Logan graphical analysis. (11)C-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model. RESULTS:Cunningham-Lassen plots indicated sigma-1R occupancies of 57 ± 2 and 85 ± 2% after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44-66%) reduction of (11)C-raclopride binding was only observed at 60 mg/kg pridopidine. CONCLUSIONS:At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D2Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity.
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Project description:Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, we examined ASH1L, the mammalian homolog of the Drosophila melanogaster Trithorax group (TrxG) protein Ash1. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. Here we demonstrate that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines we found that their recruitments occur independently of each other. Collectively, our data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes.
| S-EPMC2169421 | biostudies-literature