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BioRxiv : the Preprint Server For... Jun 2024Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a...
Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a variant of the type 1F Cascade known as type 1F-3. This Cascade variant complexes with a homodimer of the transposition protein TniQ and leverages the sequence specificity of Cascade to direct the integration activity of the heteromeric transposase tnsA/B, resulting in site-specific transposition of Tn6677. We desire to uncover the molecular details behind R Loop formation of 'Cascade-TniQ.' Due to the lack of a complete model of Cascade-TniQ available at atom-level resolution, we first build a complete model using AlphaFold V2.1. We then simulate this model via classical molecular dynamics and umbrella sampling to study an important regulatory component within Cascade-TniQ, known as the Cas8 'bundle.' Particularly, we show that this alpha helical bundle experiences a free energy barrier to its large-scale translatory motions and relative free energies of its states primarily dependent on a loop within a Cas7 subunit in Cascade-TniQ. Further, we comment on additional structural and dynamical regulatory points of Cascade-TniQ during R Loop formation, such as Cascade-TniQ backbone rigidity, and the potential role TniQ plays in regulating bundle dynamics. In summary, our outcomes provide the first all-atom dynamic representation of one of the largest CRISPR systems, with information that can contribute to understanding the mechanism of nucleic acid binding and, eventually, to transposase recruitment itself. Such information may prove informative to advance genome engineering efforts.
PubMed: 38948825
DOI: 10.1101/2024.06.21.600075 -
BioRxiv : the Preprint Server For... Jun 2024Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and...
Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and topological problems. Here we asked which topoisomerase(s) is/are the best candidate(s) for sensing TRC. Genome-wide topoisomerase binding sites were mapped in parallel for all the nuclear topoisomerases (TOP1, TOP2A, TOP2B, TOP3A and TOP3B). To increase the signal to noise ratio (SNR), we used ectopic expression of those topoisomerases in H293 cells followed by a modified CUT&Tag method. Although each topoisomerase showed distinct binding patterns, all topoisomerase binding signals positively correlated with gene transcription. TOP3A binding signals were suppressed by DNA replication inhibition. This was also observed but to a lesser extent for TOP2A and TOP2B. Hence, we propose the involvement of TOP3A in sensing both head-on TRCs (HO-TRCs) and codirectional TRCs (CD-TRCs). In which case, the TOP3A signals appear concentrated within the promoters and first 20 kb regions of the 5' -end of genes, suggesting the prevalence of TRCs and the recruitment of TOP3A in the 5'-regions of transcribed and replicated genes.
PubMed: 38948815
DOI: 10.1101/2024.06.17.599352 -
BioRxiv : the Preprint Server For... Jun 2024Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These...
UNLABELLED
Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA ) and CypA-depleted (CypA ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA cells but not in CypA cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA cells but not in CypA cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA cells had lower activity compared to those from CypA cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA and CypA cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor.
IMPORTANCE
HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.
PubMed: 38948800
DOI: 10.1101/2024.06.15.599180 -
BioRxiv : the Preprint Server For... Jun 2024Human gut species encode numerous (eight or more) tightly regulated capsular polysaccharides (CPS). Specialized paralogs of the universal transcription elongation...
Human gut species encode numerous (eight or more) tightly regulated capsular polysaccharides (CPS). Specialized paralogs of the universal transcription elongation factor NusG, called UpxY (Y), and an anti-Y UpxZ (Z) are encoded by the first two genes of each CPS operon. The Y-Z regulators combine with promoter inversions to limit CPS transcription to a single operon in most cells. Y enhances transcript elongation whereas Z inhibits noncognate Ys. How Y distinguishes among cognate CPS operons and how Z inhibits only noncognate Ys are unknown. Using in-vivo nascent-RNA sequencing and p romoter-less in v itr o transcription (PIVoT), we establish that Y recognizes a paused RNA polymerase via sequences in both the exposed non-template DNA and the upstream duplex DNA. Y association is aided by novel 'pause-then-escape' nascent RNA hairpins. Z binds non-cognate Ys to directly inhibit Y association. This Y-Z hierarchical regulatory program allows to create CPS subpopulations for optimal fitness.
PubMed: 38948710
DOI: 10.1101/2024.06.21.599965 -
BioRxiv : the Preprint Server For... Jun 2024The bacterial toxin colibactin, produced primarily by the B2 phylogroup of underlies some cases of colorectal cancers. Colibactin crosslinks DNA and induces genotoxic...
UNLABELLED
The bacterial toxin colibactin, produced primarily by the B2 phylogroup of underlies some cases of colorectal cancers. Colibactin crosslinks DNA and induces genotoxic damage in both mammalian and bacterial cells. While the mechanisms facilitating colibactin delivery remain unclear, results from multiple studies supported a delivery model that necessitates cell-cell contact. We directly tested this requirement in bacterial cultures by monitoring the spatiotemporal dynamics of the DNA damage response using a fluorescent transcriptional reporter. We found that in mixed-cell populations, DNA damage saturated within twelve hours and was detectable even in reporter cells separated from colibactin producers by hundreds of microns. Experiments with distinctly separated producer and reporter colonies revealed that the intensity of DNA damage decays similarly with distance regardless of colony contact. Our work reveals that cell contact is inconsequential for colibactin delivery in bacteria and suggests that contact-dependence needs to be reexamined in mammalian cells as well.
IMPORTANCE
Colibactin is a bacteria-produced toxin that binds and damages DNA. It has been widely studied in mammalian cells due to its potential role in tumorigenesis. However, fundamental questions about its impact in bacteria remain underexplored. We used as a model system to study colibactin toxicity in neighboring bacteria and directly tested if cell-cell contact is required for toxicity, as has previously been proposed. We found that colibactin can induce DNA damage in bacteria hundreds of microns away and that the intensity of DNA damage presents similarly regardless of cell-cell contact. Our work further suggests that the requirement for cell-cell contact for colibactin-induced toxicity also needs to be reevaluated in mammalian cells.
PubMed: 38948699
DOI: 10.1101/2024.06.21.600066 -
MLife Jun 2024Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is...
Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.
PubMed: 38948148
DOI: 10.1002/mlf2.12119 -
IScience Jun 2024The NAD-dependent deacetylase SIRT7 is a pivotal regulator of DNA damage response (DDR) and a promising drug target for developing cancer therapeutics. However, limited...
The NAD-dependent deacetylase SIRT7 is a pivotal regulator of DNA damage response (DDR) and a promising drug target for developing cancer therapeutics. However, limited progress has been made in SIRT7 modulator discovery. Here, we applied peptide-based deacetylase platforms for SIRT7 enzymatic evaluation and successfully identified a potent SIRT7 inhibitor . We initially isolated bioactive from cockroach () extracts and then developed the synthesis of this compound Further investigation revealed that impaired SIRT7 enzymatic activities through occupation of the NAD binding pocket. attenuated DNA damage repair induced by ionizing radiation (IR) in colorectal cancer cells and exhibited a synergistic anticancer effect when used in combination with etoposide. Overall, our study not only identified as a selective SIRT7 inhibitor from insect resources, but also confirmed its potential use in combined chemo-radiotherapy by interfering in the DNA damage repair process.
PubMed: 38947512
DOI: 10.1016/j.isci.2024.110014 -
Journal of Cancer 2024In the realm of cancer research, particularly hepatocellular carcinoma (HCC), TAR DNA-binding protein (TARDBP) has transitioned from being associated with...
In the realm of cancer research, particularly hepatocellular carcinoma (HCC), TAR DNA-binding protein (TARDBP) has transitioned from being associated with neurodegenerative diseases to emerging as a significant molecule in oncology due to its aberrant expression in HCC and other malignancies. This shift underlines the versatility of TARDBP and its critical role in tumorigenesis. Our study illuminates TARDBP's universal upregulation across various cancers, indicating its involvement in fundamental oncogenic processes and potential impact on genomic instability. The relationship between TARDBP expression and tumor mutational burden (TMB) across several cancers highlights its influence on a key hallmark of cancer progression. Additionally, TARDBP's interaction with immune and inflammatory factors within the tumor microenvironment, including its association with immune-stimulatory factors and inverse relationship with immune inhibitors, suggests its role in modulating immune evasion. Clinically, TARDBP's aberrant expression correlates with adverse patient outcomes in HCC, making it a promising candidate for therapeutic targeting. The study concludes that TARDBP holds significant potential as a novel therapeutic target in HCC and possibly other malignancies, meriting further exploration to integrate TARDBP-targeted therapies into cancer treatment protocols, thereby advancing the field of precision medicine.
PubMed: 38947395
DOI: 10.7150/jca.96800 -
ACS Central Science Jun 2024Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to...
Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes. Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically acetylated and fluorescently tagged analogs. The resulting synthetic analogs were employed to decipher the molecular role of Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31 and Lys-57 significantly inhibit the DNA binding activity of Max. Furthermore, by utilizing high-throughput binding measurements, we assessed the binding activities of the modified Max variants across diverse DNA sequences. Our results indicate that acetylation marks can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.
PubMed: 38947213
DOI: 10.1021/acscentsci.4c00686 -
Journal of Extracellular Biology Jun 2024Chemoresistance is a common problem in ovarian cancer (OvCa) treatment, where resistant cells, in response to chemotherapy, secrete small extracellular vesicles (sEVs),...
Chemoresistance is a common problem in ovarian cancer (OvCa) treatment, where resistant cells, in response to chemotherapy, secrete small extracellular vesicles (sEVs), known as chemo-sEVs, that transfer resistance to recipient cells. sEVs are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs), whose trafficking is regulated by Ras-associated binding (RAB) GTPases that mediate sEVs secretion or lysosomal degradation. A decrease in lysosomal function can promote sEVs secretion, but the relationship between MVEs trafficking pathways and sEVs secretion in OvCa chemoresistance is unclear. Here, we show that A2780cis cisplatin (CCDP) resistant OvCa cells had an increased number of MVEs and ILVs structures, higher levels of Endosomal Sorting Complex Required for Transport (ESCRTs) machinery components, and RAB27A compared to A2780 CDDP-sensitive OvCa cells. CDDP promoted the secretion of chemo-sEVs in A2780cis cells, enriched in DNA damage response proteins. A2780cis cells exhibited poor lysosomal function with reduced levels of RAB7, essential in MVEs-Lysosomal trafficking. The silencing of RAB27A in A2780cis cells prevents the Chemo-EVs secretion, reduces its chemoresistance and restores lysosomal function and levels of RAB7, switching them into an A2780-like cellular phenotype. Enhancing lysosomal function with rapamycin reduced chemo-sEVs secretion. Our results suggest that adjusting the balance between secretory MVEs and lysosomal MVEs trafficking could be a promising strategy for overcoming CDDP chemoresistance in OvCa.
PubMed: 38947172
DOI: 10.1002/jex2.157