Genome Replication Is Associated With Release of Immunogenic DNA Waste
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ABSTRACT:
SUBMITTER: Schubert N
PROVIDER: S-EPMC9130835 | biostudies-literature | 2022 Jan
REPOSITORIES: biostudies-literature
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Project description:Plasmodium species have variable genome compositions: many have an A/T content >80%, while others are similar in composition to human cells. Here, we made a direct comparison of DNA replication dynamics in two Plasmodium species whose genomes differ by ∼20% A/T content. This yielded fundamental insights into how DNA composition may affect replication. The highly A/T-biased genome of Plasmodium falciparum showed unusual replication dynamics that were not observed in the more balanced Plasmodium knowlesi-which had dynamics more like those of human cell lines. Replication forks moved 50% slower in P. falciparum than in P. knowlesi. In P. falciparum, replication forks slowed down over the course of S-phase, whereas in P. knowlesi, fork speed increased as in human cells. Furthermore, in both P. knowlesi and human cells, replication forks were strikingly slowed by sequences of particularly high A/T bias, but in P. falciparum, although replication forks were inherently slow, they were not particularly slow in such biased sequences. Thus, the replisome of P. falciparum may have evolved alongside its extremely biased genome, making it unusually robust to sequence bias. Since several antimalarial drugs act to stall DNA replication, this study may have implications for the effectiveness of, and development of, antimalarial therapies.
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Project description:IntroductionImmunogenic cell death (ICD) has emerged as a novel option for cancer immunotherapy. The key determinants of ICD encompass antigenicity (the presence of antigens) and adjuvanticity, which involves the release of damage-associated molecular patterns (DAMPs) and various cytokines and chemokines. CX3CL1, also known as neurotactin or fractalkine, is a chemokine involved in cellular signalling and immune cell interactions. CX3CL1 has been denoted as a "find me" signal that stimulates chemotaxis of immune cells towards dying cells, facilitating efferocytosis and antigen presentation. However, in the context of ICD, it is uncertain whether CX3CL1 is an important mediator of the effects of ICD.MethodsIn this study, we investigated the intricate role of CX3CL1 in immunogenic apoptosis induced by mitoxantrone (MTX) in cancer cells. The Luminex xMAP technology was used to quantify murine cytokines, chemokines and growth factors to identify pivotal regulatory cytokines released by murine fibrosarcoma MCA205 and melanoma B16-F10 cells undergoing ICD. Moreover, a murine tumour prophylactic vaccination model was employed to analyse the effect of CX3CL1 on the activation of an adaptive immune response against MCA205 cells undergoing ICD. Furthermore, thorough analysis of the TCGA-SKCM public dataset from 98 melanoma patients revealed the role of CX3CL1 and its receptor CX3CR1 in melanoma patients.ResultsOur findings demonstrate enhanced CX3CL1 release from apoptotic MCA205 and B16-F10 cells (regardless of the cell type) but not if they are undergoing ferroptosis or accidental necrosis. Moreover, the addition of recombinant CX3CL1 to non-immunogenic doses of MTX-treated, apoptotically dying cancer cells in the murine prophylactic tumour vaccination model induced a robust immunogenic response, effectively increasing the survival of the mice. Furthermore, analysis of melanoma patient data revealed enhanced survival rates in individuals exhibiting elevated levels of CD8+ T cells expressing CX3CR1.ConclusionThese data collectively underscore the importance of the release of CX3CL1 in eliciting an immunogenic response against dying cancer cells and suggest that CX3CL1 may serve as a key switch in conferring immunogenicity to apoptosis.
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Project description:Dengue virus (DENV) infection remains a challenging health threat worldwide. Ubiquitin-specific protease 18 (USP18), which preserves the anti-interferon (IFN) effect, is an ideal target through which DENV mediates its own immune evasion. However, much of the function and mechanism of USP18 in regulating DENV replication remains incompletely understood. In addition, whether USP18 regulates DENV replication merely by causing IFN hyporesponsiveness is not clear. In the present study, by using several different approaches to block IFN signaling, including IFN neutralizing antibodies (Abs), anti-IFN receptor Abs, Janus kinase inhibitors and IFN alpha and beta receptor subunit 1 (IFNAR1)knockout cells, we showed that USP18 may regulate DENV replication in IFN-associated and IFN-unassociated manners. Localized in mitochondria, USP18 regulated the release of mitochondrial DNA (mtDNA) to the cytosol to affect viral replication, and mechanisms such as mitochondrial reactive oxygen species (mtROS) production, changes in mitochondrial membrane potential, mobilization of calcium into mitochondria, 8-oxoguanine DNA glycosylase 1 (OGG1) expression, oxidation and fragmentation of mtDNA, and opening of the mitochondrial permeability transition pore (mPTP) were involved in USP18-regulated mtDNA release to the cytosol. We therefore identify mitochondrial machineries that are regulated by USP18 to affect DENV replication and its association with IFN effects.
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Project description:Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands. However, coupled replication presents a largely unrecognized topological problem. Because DNA polymerase must travel a helical path during synthesis, the physical connection between leading- and lagging-strand polymerases causes the daughter strands to entwine, or produces extensive build-up of negative supercoils in the newly synthesized DNA. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is unknown. Here we examine the dynamics of the Escherichia coli replisome, using ensemble and single-molecule methods, and show that the replisome may solve the topological problem independent of topoisomerases. We find that the lagging-strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome because of its contact with the leading-strand polymerase. This behaviour, referred to as 'signal release', had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments. However, we observe that signal release is independent of primase and does not seem to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.
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Project description:Cancer stem cells (CSCs) are a major cause of tumor therapy failure. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies have shown that functional DNA repair via homologous recombination (HR) prevents radiation-induced accumulation of DNA in the cytoplasm, thereby inhibiting the intracellular immune response. However, it is unclear whether CSCs can suppress radiation-induced cytoplasmic dsDNA formation. Here, we show that the increased radioresistance of ALDH1-positive breast cancer stem cells (BCSCs) in S phase is mediated by both enhanced DNA double-strand break repair and improved replication fork protection due to HR. Both HR-mediated processes lead to suppression of radiation-induced replication stress and consequently reduction of cytoplasmic dsDNA. The amount of cytoplasmic dsDNA correlated significantly with BCSC content (p=0.0002). This clearly indicates that HR-dependent avoidance of radiation-induced replication stress mediates radioresistance and contributes to its immune evasion. Consistent with this, enhancement of replication stress by inhibition of ataxia telangiectasia and RAD3 related (ATR) resulted in significant radiosensitization (SER37 increase 1.7-2.8 Gy, p<0.0001). Therefore, disruption of HR-mediated processes, particularly in replication, opens a CSC-specific radiosensitization option by enhancing their intracellular immune response.
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Project description:The eukaryotic genome is in a constant state of modification and repair. Faithful transmission of the genomic information from parent to daughter cells depends upon an extensive system of surveillance, signaling, and DNA repair, as well as accurate synthesis of DNA during replication. Often, replicative synthesis occurs over regions of DNA that have not yet been repaired, presenting further challenges to genomic stability. DNA polymerase δ (pol δ) occupies a central role in all of these processes: catalyzing the accurate replication of a majority of the genome, participating in several DNA repair synthetic pathways, and contributing structurally to the accurate bypass of problematic lesions during translesion synthesis. The concerted actions of pol δ on the lagging strand, pol ϵ on the leading strand, associated replicative factors, and the mismatch repair (MMR) proteins results in a mutation rate of less than one misincorporation per genome per replication cycle. This low mutation rate provides a high level of protection against genetic defects during development and may prevent the initiation of malignancies in somatic cells. This review explores the role of pol δ in replication fidelity and genome maintenance.
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Project description:Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4BC74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4BC74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment.Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short.
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Project description:DNA polymerase iota (Pol ι) is a Y-family lesion bypass polymerases, characterized by its remarkably low fidelity. While Pol ι is ubiquitously expressed in nearly all cell types, its function remains largely undefined. Previous studies in cellular and mouse models deficient in Pol ι have revealed no discernible phenotype. In this report, we show that Pol ι plays an important role in mitotic DNA synthesis. Cells lacking Pol ι exhibit heightened vulnerability to replication stress, increased formation of micronuclei, and accumulation of chromosomal breaks. The protein level of Pol ι is tightly regulated and restricted to G2/M phase cells via CRL7/FBXW11-mediated proteasomal degradation. The absence of Pol ι during S phase ensures that its error-prone DNA synthesis activity is excluded from interfering with normal DNA replication. These findings suggest that Pol ι function is critical in resolving DNA damage-induced replication stress prior to the completion of mitosis.
2025-07-01 | PXD065672 |
Project description:Genome integrity requires faithful chromosome duplication. Origins of replication, the genomic sites at which DNA replication initiates, are scattered throughout the genome. Their mapping at a genomic scale in multicellular organisms has been challenging. In this study we profiled origins in Arabidopsis thaliana by high-throughput sequencing of newly synthesized DNA and identified ~1,500 putative origins genome-wide. This was supported by chromatin immunoprecipitation and microarray (ChIP-chip) experiments to identify ORC1- and CDC6-binding sites. We validated origin activity independently by measuring the abundance of nascent DNA strands. The midpoints of most A. thaliana origin regions are preferentially located within the 5' half of genes, enriched in G+C, histone H2A.Z, H3K4me2, H3K4me3 and H4K5ac, and depleted in H3K4me1 and H3K9me2. Our data help clarify the epigenetic specification of DNA replication origins in A. thaliana and have implications for other eukaryotes.
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Project description:Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins' structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5-3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks.
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