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Journal of Molecular Biology Jun 2024CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair....
CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.
PubMed: 38908783
DOI: 10.1016/j.jmb.2024.168672 -
Cell Genomics Jun 2024The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important...
The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.
PubMed: 38908378
DOI: 10.1016/j.xgen.2024.100590 -
STAR Protocols Jun 2024Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions...
Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts. For complete details on the use and execution of this protocol, please refer to Zhao et al..
PubMed: 38907997
DOI: 10.1016/j.xpro.2024.103149 -
Life Science Alliance Sep 2024H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found...
H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 toward phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.
Topics: Histones; Tumor Suppressor Protein p53; Cell Nucleus; Humans; Enhancer of Zeste Homolog 2 Protein; G1 Phase; S Phase; Nuclear Envelope; Methylation; Animals; Nucleosomes
PubMed: 38906678
DOI: 10.26508/lsa.202402835 -
Life Science Alliance Sep 2024Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage....
Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage. Although they are already applied against myeloid cancers, important aspects of their mode of action remain unknown, highly limiting their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA damage. Under low DNMT activity, only decitabine has a substantial impact. Conversely, when DNMT activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA hypomethylation or RNA-associated processes. By investigating proteome dynamics on chromatin, we show that decitabine induces a strictly DNMT-dependent multifaceted DNA damage response based on chromatin recruitment, but not expression-level changes of repair-associated proteins. The choice of DNA repair pathway hereby depends on the severity of decitabine-induced DNA lesions. Although under moderate DNMT activity, mismatch (MMR), base excision (BER), and Fanconi anaemia-dependent DNA repair combined with homologous recombination are activated in response to decitabine, high DNMT activity and therefore immense replication stress induce activation of MMR and BER followed by non-homologous end joining.
Topics: Decitabine; DNA Damage; Humans; DNA Repair; DNA Methylation; Azacitidine; Antimetabolites, Antineoplastic; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferases; Chromatin; DNA Modification Methylases
PubMed: 38906675
DOI: 10.26508/lsa.202302437 -
European Journal of Pharmaceutical... Jun 2024The hepatitis B virus (HBV) capsid or core protein is a promising drug target currently being investigated for potential curative therapies for chronic HBV infection. In...
The hepatitis B virus (HBV) capsid or core protein is a promising drug target currently being investigated for potential curative therapies for chronic HBV infection. In this study, we performed extensive in vitro and in vivo characterization of a novel and potent HBV core protein assembly modulator (CpAM), CU15, for both anti-HBV activity and druggability properties. CU15 potently inhibited HBV DNA replication in in vitro HBV-infected HepG2.2.15 cells (EC of 8.6 nM), with a low serum shift. It was also effective in inhibiting HBV DNA and cccDNA formation in de novo HBV-infected primary human hepatocytes. Furthermore, CU15 was active across several HBV genotypes and across clinically relevant core protein variants. After oral administration to an in vivo HBV mouse model, CU15 significantly reduced plasma HBV DNA and RNA levels, at plasma exposure consistent with the estimated in vitro potency. In vitro, CU15 exhibited excellent passive permeability and relatively high metabolic stability in liver preparations across species (human > dog> rat). In vitro human liver microsomal studies suggest that the compound's major metabolic pathway is CYP3A-mediated oxidation. Consistent with the in vitro findings, CU15 is a compound with a low-to-moderate clearance and high oral bioavailability in rats and dogs. Based on the apparent in vitro-in vivo correlation observed, CU15 has the potential to exhibit low clearance and high oral bioavailability in humans. In addition, CU15 also showed low drug-drug interaction liability with an acceptable in vitro safety profile (IC > 10 µM).
PubMed: 38906232
DOI: 10.1016/j.ejps.2024.106834 -
Marine Genomics Jun 2024Environmental DNA (eDNA) analyses of species present in marine environments is the most effective biological diversity measurement tool currently available. eDNA...
Environmental DNA (eDNA) analyses of species present in marine environments is the most effective biological diversity measurement tool currently available. eDNA sampling methods are an intrinsically important part of the eDNA biodiversity analysis process. Identification and development of eDNA sampling methods that are as rapid, affordable, versatile and practical as possible will improve rates of detection of marine species. Optimal outcomes of eDNA biodiversity surveys come from studies employing high levels of sampling replication, so any methods that make sampling faster and cheaper will improve scientific outcomes. eDNA sampling methods that can be applied more widely will also enable sampling from a greater range of marine surface micro-habitats, resulting in detection of a wider range of organisms. In this study, we compared diversity detection by several methods for sampling eDNA from submerged marine surfaces: polyurethane foam, nylon swabs, microfibre paint rollers, and sediment scoops. All of the methods produced a diverse range of species identifications, with >250 multicellular species represented by eDNA at the study site. We found that widely-available small paint rollers were an effective, readily available and affordable method for sampling eDNA from underwater marine surfaces. This approach enables the sampling of marine eDNA using extended poles, or potentially by remotely operated vehicles, where surface sampling by hand is impractical.
PubMed: 38905943
DOI: 10.1016/j.margen.2024.101127 -
PloS One 2024Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the...
Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome.
Topics: Schizosaccharomyces; Schizosaccharomyces pombe Proteins; DNA Replication; DNA Helicases; DNA Damage; Hydroxyurea; Stress, Physiological; DNA, Ribosomal; Chromosomal Instability
PubMed: 38905307
DOI: 10.1371/journal.pone.0300434 -
MSystems Jun 2024In nature, bacteria often survive in a stationary state with low metabolic activity. Phages use the metabolic machinery of the host cell to replicate, and, therefore,...
In nature, bacteria often survive in a stationary state with low metabolic activity. Phages use the metabolic machinery of the host cell to replicate, and, therefore, their efficacy against non-dividing cells is usually limited. Nevertheless, it was previously shown that the phage SEP1 has the remarkable capacity to actively replicate in stationary-phase cells, reducing their numbers. Here, we studied for the first time the transcriptomic profiles of both exponential and stationary cells infected with SEP1 phage using RNA-seq to gain a better understanding of this rare phenomenon. We showed that SEP1 successfully takes over the transcriptional apparatus of both exponential and stationary cells. Infection was, however, delayed in stationary cells, with genes within the module putatively implicated in host takeover. responded to SEP1 infection by upregulating three genes involved in a DNA modification system, with this being observed already 5 min after infection in exponential cells and later in stationary cells. In stationary cells, a significant number of genes involved in translation and RNA metabolic and biosynthetic processes were upregulated after 15 and 30 min of SEP1 infection in comparison with the uninfected control, showing that SEP1 activates metabolic and biosynthetic pathways necessary to its successful replication.IMPORTANCEMost phage-host interaction studies are performed with exponentially growing cells. However, this cell state is not representative of what happens in natural environments. Additionally, most phages fail to replicate in stationary cells. The phage SEP1 is one of the few phages reported to date to be able to infect stationary cells. Here, we unveiled the interaction of SEP1 with its host in both exponential and stationary states of growth at the transcriptomic level. The findings of this study provide valuable insights for a better implementation of phage therapy since phages able to infect stationary cells could be more efficient in the treatment of recalcitrant infections.
PubMed: 38904376
DOI: 10.1128/msystems.00263-24 -
F1000Research 2023Paediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is...
BACKGROUND
Paediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) can access the nervous system and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma.
METHODS
Here, we perform extensive data mining, integration and re-analysis of ZIKV infection datasets to highlight molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We collate infection data of multiple neuroblastoma cell lines by different ZIKV strains from a body of published literature to inform the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating published transcriptomics, interaction proteomics, dependency factor and compound datasets we propose the involvement of multiple host systems during ZIKV infection.
RESULTS
Through data mining of published literature, we observed most paediatric neuroblastoma cell lines to be highly susceptible to ZIKV infection and propose the PRVABC59 ZIKV strain to be the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV infection is dependent on sterol regulatory element binding protein (SREBP)-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV non-structural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon.
CONCLUSIONS
Our work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials.
Topics: Humans; Neuroblastoma; Oncolytic Virotherapy; Zika Virus; Proteomics; Cell Line, Tumor; Zika Virus Infection; Transcriptome
PubMed: 38903860
DOI: 10.12688/f1000research.132627.3