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Genes Mar 2024The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation... (Review)
Review
The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.
Topics: Humans; DNA; DNA Replication; DNA Polymerase III; Minichromosome Maintenance Proteins; Genomic Instability
PubMed: 38540419
DOI: 10.3390/genes15030360 -
BioRxiv : the Preprint Server For... Nov 2023There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood....
There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, "a site to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed . We conclude that, if sites at which Mcm double-hexamers are loaded can function as replication origins, then DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.
PubMed: 38014147
DOI: 10.1101/2023.04.11.536402 -
PRX Life 2023Balanced biosynthesis is the hallmark of bacterial cell physiology, where the concentrations of stable proteins remain steady. However, this poses a conceptual challenge...
Balanced biosynthesis is the hallmark of bacterial cell physiology, where the concentrations of stable proteins remain steady. However, this poses a conceptual challenge to modeling the cell-cycle and cell-size controls in bacteria, as prevailing concentration-based eukaryote models are not directly applicable. In this study, we revisit and significantly extend the initiator-titration model, proposed 30 years ago, and we explain how bacteria precisely and robustly control replication initiation based on the mechanism of protein copy-number sensing. Using a mean-field approach, we first derive an analytical expression of the cell size at initiation based on three biological mechanistic control parameters for an extended initiator-titration model. We also study the stability of our model analytically and show that initiation can become unstable in multifork replication conditions. Using simulations, we further show that the presence of the conversion between active and inactive initiator protein forms significantly represses initiation instability. Importantly, the two-step Poisson process set by the initiator titration step results in significantly improved initiation synchrony with scaling rather than the standard scaling in the Poisson process, where is the total number of initiators required for initiation. Our results answer two long-standing questions in replication initiation: (i) Why do bacteria produce almost two orders of magnitude more DnaA, the master initiator proteins, than required for initiation? (ii) Why does DnaA exist in active (DnaA-ATP) and inactive (DnaA-ADP) forms if only the active form is competent for initiation? The mechanism presented in this work provides a satisfying general solution to how the cell can achieve precision control without sensing protein concentrations, with broad implications from evolution to the design of synthetic cells.
PubMed: 38550259
DOI: 10.1103/prxlife.1.013011 -
Nature Communications Mar 2024The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and...
The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.
Topics: Protein Kinases; Phosphorylation; Signal Transduction; Replication Protein A; Ataxia Telangiectasia Mutated Proteins; DNA-Binding Proteins; DNA Replication; DNA Damage; DNA, Single-Stranded; DNA Repair
PubMed: 38472229
DOI: 10.1038/s41467-024-46578-0 -
Proceedings of the National Academy of... Jan 2024High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased...
High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.
Topics: Humans; Mice; Animals; Anaplastic Lymphoma Kinase; Receptor Protein-Tyrosine Kinases; Cell Proliferation; Cell Line, Tumor; Neuroblastoma; DNA Repair; DNA Damage; Ataxia Telangiectasia Mutated Proteins
PubMed: 38154064
DOI: 10.1073/pnas.2315242121 -
Cells Nov 2023In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication, and repair....
In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication, and repair. Our previous studies have demonstrated that SIRT1 binds replication origins and inhibits replication initiation from a group of potential initiation sites (dormant origins). We studied the effects of aging and SIRT1 activity on replication origin usage and the incidence of transcription-replication collisions (creating R-loop structures) in adult human cells obtained at different time points during chronological aging and in cancer cells. In primary, untransformed cells, SIRT1 activity declined and the prevalence of R-loops rose with chronological aging. Both the reduction in SIRT1 activity and the increased abundance of R-loops were also observed during the passage of primary cells in culture. All cells, regardless of donor age or transformation status, reacted to the short-term, acute chemical inhibition of SIRT1 with the activation of excessive replication initiation events coincident with an increased prevalence of R-loops. However, cancer cells activated dormant replication origins, genome-wide, during long-term proliferation with mutated or depleted SIRT1, whereas, in primary cells, the aging-associated SIRT1-mediated activation of dormant origins was restricted to rDNA loci. These observations suggest that chronological aging and the associated decline in SIRT1 activity relax the regulatory networks that protect cells against excess replication and that the mechanisms protecting from replication-transcription collisions at the rDNA loci manifest as differentially enhanced sensitivities to SIRT1 decline and chronological aging.
Topics: Humans; DNA, Ribosomal; R-Loop Structures; Sirtuin 1; DNA Replication; Aging
PubMed: 37998365
DOI: 10.3390/cells12222630 -
Advances in Nutrition (Bethesda, Md.) Sep 2023Cellular senescence has long been considered a permanent state of cell cycle arrest occurring in proliferating cells subject to different stressors, used as a cellular... (Review)
Review
Cellular senescence has long been considered a permanent state of cell cycle arrest occurring in proliferating cells subject to different stressors, used as a cellular defense mechanism from acquiring potentially harmful genetic faults. However, recent studies highlight that senescent cells might also alter the local tissue environment and concur to chronic inflammation and cancer risk by secreting inflammatory and matrix remodeling factors, acquiring a senescence-associated secretory phenotype (SASP). Indeed, during aging and age-related diseases, senescent cells amass in mammalian tissues, likely contributing to the inevitable loss of tissue function as we age. Cellular senescence has thus become one potential target to tackle age-associated diseases as well as cancer development. One important aspect characterizing senescent cells is their telomere length. Telomeres shorten as a consequence of multiple cellular replications, gradually leading to permanent cell cycle arrest, known as replicative senescence. Interestingly, in the large majority of cancer cells, a senescence escape strategy is used and telomere length is maintained by telomerase, thus favoring cancer initiation and tumor survival. There is growing evidence showing how (poly)phenols can impact telomere maintenance through different molecular mechanisms depending on dose and cell phenotypes. Although normally, (poly)phenols maintain telomere length and support telomerase activity, in cancer cells this activity is negatively modulated, thus accelerating telomere attrition and promoting cancer cell death. Some (poly)phenols have also been shown to exert senolytic activity, thus suggesting both antiaging (directly eliminating senescent cells) and anticancer (indirectly, via SASP inhibition) potentials. In this review, we analyze selective (poly)phenol mechanisms in senescent and cancer cells to discriminate between in vitro and in vivo evidence and human applications considering (poly)phenol bioavailability, the influence of the gut microbiota, and their dose-response effects.
Topics: Animals; Humans; Telomerase; Phenols; Cell Survival; Phenol; Aging; Neoplasms; Cell Proliferation; Mammals
PubMed: 37271484
DOI: 10.1016/j.advnut.2023.05.014 -
Frontiers in Immunology 2023Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective...
BACKGROUND
Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.
METHODS
By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.
RESULTS
We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.
CONCLUSION
Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.
Topics: Animals; Salmonidae; CRISPR-Cas Systems; Signal Transduction; Cell Line; Salmon; Infectious pancreatic necrosis virus; Mammals
PubMed: 37588594
DOI: 10.3389/fimmu.2023.1214912 -
Experimental and Therapeutic Medicine Jul 2024Mycoplasmas, the smallest self-replicating prokaryotes without a cell wall, are the most prevalent and extensively studied species in humans. They significantly... (Review)
Review
Mycoplasmas, the smallest self-replicating prokaryotes without a cell wall, are the most prevalent and extensively studied species in humans. They significantly contribute to chronic respiratory tract illnesses and pneumonia, with children and adolescents being particularly vulnerable. () infections typically tend to be self-limiting and mild but can progress to severe or even life-threatening conditions in certain individuals. Extrapulmonary effects often occur without pneumonia, and both intrapulmonary and extrapulmonary complications operate through separate pathological mechanisms. The indirect immune-mediated damage of the immune system, vascular blockages brought on by vasculitis or thrombosis and direct harm from invasion or locally induced inflammatory cytokines are potential causes of extrapulmonary manifestations due to . Proteins associated with adhesion serve as the primary factor crucial for the pathogenicity of , relying on a specialized polarized terminal attachment organelle. The type and density of these host receptors significantly impact the adhesion and movement of , subsequently influencing the pathogenic mechanism and infection outcomes. Adjacent proteins are crucial for the proper assembly of the attachment organelle, with variations in the genetic domains of P1, P40 and P90 surfaces contributing to the variability of clinical symptoms and offering new avenues for developing vaccines against infections. causes oxidative stress within respiratory tract epithelial cells by adhering to host cells and releasing hydrogen peroxide and superoxide radicals. This oxidative stress enhances the vulnerability of host cells to harm induced by oxygen molecules. The lack of superoxide dismutase and catalase of bacteria allows it to hinder the catalase activity of the host cell, leading to the reduced breakdown of peroxides. Lung macrophages play a significant role in managing infection, identifying it via Toll-like receptor 2 and initiating the myeloid differentiation primary response gene 88-nuclear factor κΒ signaling cascade. However, the precise mechanisms enabling to evade intracellular host defenses remain unknown, necessitating further exploration of the pathways involved in intracellular survival. The present comprehensive review delves into the pathogenesis of infection within the pulmonary system and into extrapulmonary areas, outlining its impact.
PubMed: 38765654
DOI: 10.3892/etm.2024.12559 -
Prevention Science : the Official... Oct 2023Despite evidence that pre-exposure prophylaxis (PrEP) reduces HIV risk, initiation and adherence remain low among vulnerable communities. Motivational interviewing (MI)... (Review)
Review
Despite evidence that pre-exposure prophylaxis (PrEP) reduces HIV risk, initiation and adherence remain low among vulnerable communities. Motivational interviewing (MI) can improve HIV prevention behaviors. However, limited research identifies how MI impacts PrEP uptake and adherence. This scoping review examines essential components of MI-based interventions that aimed to improve PrEP use, including the number and duration of sessions, counselor characteristics, and interview content. We searched four databases, PubMed, CINAHL Plus, Embase, and Web of Science, and reviewed 379 articles. Studies were considered if they (a) were published between 2012 and 2023, (b) used MI independently or part of a multi-component intervention strategy, and (c) focused on improving PrEP initiation or adherence. Seven articles met inclusion criteria. Regarding intervention components, the number of MI sessions varied and duration ranged between 15 and 60 min. MI counselors varied in credentialing and demographic characteristics. MI content included PrEP education, identifying initiation and adherence barriers, and strategizing ways to overcome barriers. MI is an important component of interventions that aim to improve PrEP initiation and adherence. However, the variability and limited details across studies hinder our ability to assess MI efficacy on PrEP initiation and adherence or replicate these approaches in future interventions.
Topics: Humans; Male; Motivational Interviewing; HIV Infections; Acquired Immunodeficiency Syndrome; Pre-Exposure Prophylaxis; Cognition; Homosexuality, Male
PubMed: 37249729
DOI: 10.1007/s11121-023-01554-w