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Science (New York, N.Y.) Sep 2023CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies...
CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.
Topics: Animals; Humans; Mice; Cell Cycle Proteins; DNA Replication; DNA-Binding Proteins; Minichromosome Maintenance Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Protein Interaction Mapping; Computer Simulation; Nuclear Proteins; Dwarfism; Microcephaly; Xenopus laevis
PubMed: 37590370
DOI: 10.1126/science.adi3448 -
Nature Reviews. Molecular Cell Biology Feb 2024The expression of mitochondrial genes is regulated in response to the metabolic needs of different cell types, but the basic mechanisms underlying this process are still... (Review)
Review
The expression of mitochondrial genes is regulated in response to the metabolic needs of different cell types, but the basic mechanisms underlying this process are still poorly understood. In this Review, we describe how different layers of regulation cooperate to fine tune initiation of both mitochondrial DNA (mtDNA) transcription and replication in human cells. We discuss our current understanding of the molecular mechanisms that drive and regulate transcription initiation from mtDNA promoters, and how the packaging of mtDNA into nucleoids can control the number of mtDNA molecules available for both transcription and replication. Indeed, a unique aspect of the mitochondrial transcription machinery is that it is coupled to mtDNA replication, such that mitochondrial RNA polymerase is additionally required for primer synthesis at mtDNA origins of replication. We discuss how the choice between replication-primer formation and genome-length RNA synthesis is controlled at the main origin of replication (OriH) and how the recent discovery of an additional mitochondrial promoter (LSP2) in humans may change this long-standing model.
Topics: Humans; Transcription, Genetic; DNA Replication; DNA, Mitochondrial; Mitochondria; DNA-Directed RNA Polymerases; Mitochondrial Proteins
PubMed: 37783784
DOI: 10.1038/s41580-023-00661-4 -
Nature Genetics Aug 2023Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of...
Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis.
Topics: Humans; DNA-Binding Proteins; Cell Cycle Proteins; DNA Replication; DNA Damage; Oncogenes; Carcinogenesis; Cohesins
PubMed: 37500731
DOI: 10.1038/s41588-023-01458-z -
Cell Reports Jul 2023The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct...
The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct assays to analyze ATR functions at ongoing and newly assembled replication forks upon replication inhibition by hydroxyurea. At ongoing forks, ATR inhibitor (ATRi) increases MRE11- and EXO1-mediated nascent DNA degradation from PrimPol-generated, single-stranded DNA (ssDNA) gaps. ATRi also exposes template ssDNA through fork uncoupling and nascent DNA degradation. Electron microscopy reveals that ATRi reduces reversed forks by increasing gap-dependent nascent DNA degradation. At new forks, ATRi triggers MRE11- and CtIP-initiated template DNA degradation by EXO1, exposing nascent ssDNA. Upon PARP inhibition, ATRi preferentially exacerbates gap-dependent nascent DNA degradation at ongoing forks in BRCA1/2-deficient cells and disrupts the restored gap protection in BRCA1-deficient, PARP-inhibitor-resistant cells. Thus, ATR protects ongoing and new forks through distinct mechanisms, providing an extended view of ATR's functions in stabilizing replication forks.
Topics: BRCA1 Protein; BRCA2 Protein; DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Poly(ADP-ribose) Polymerase Inhibitors; Humans; Ataxia Telangiectasia Mutated Proteins
PubMed: 37454295
DOI: 10.1016/j.celrep.2023.112792 -
Immunity Nov 2023Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm...
Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm and initiates innate immune responses and has been implicated in various infectious and non-infectious contexts. cGAS binds to the backbone of dsDNA and generates the second messenger, cGAMP, which activates the stimulator of interferon genes (STING). Here, we show that the endogenous polyamines spermine and spermidine attenuated cGAS activity and innate immune responses. Mechanistically, spermine and spermidine induced the transition of B-form DNA to Z-form DNA (Z-DNA), thereby decreasing its binding affinity with cGAS. Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism that decreases the cellular concentrations of spermine and spermidine, enhanced cGAS activation by inhibiting cellular Z-DNA accumulation; SAT1 deficiency promoted herpes simplex virus 1 (HSV-1) replication in vivo. The results indicate that spermine and spermidine induce dsDNA to adopt the Z-form conformation and that SAT1-mediated polyamine metabolism orchestrates cGAS activity.
Topics: Spermine; Spermidine; DNA, Z-Form; DNA, B-Form; DNA; Nucleotidyltransferases; Polyamines; Immunity, Innate
PubMed: 37848037
DOI: 10.1016/j.immuni.2023.09.012 -
Molecular Cell Nov 2023Cellular senescence refers to an irreversible state of cell-cycle arrest and plays important roles in aging and cancer biology. Because senescence is associated with...
Cellular senescence refers to an irreversible state of cell-cycle arrest and plays important roles in aging and cancer biology. Because senescence is associated with increased cell size, we used reversible cell-cycle arrests combined with growth rate modulation to study how excessive growth affects proliferation. We find that enlarged cells upregulate p21, which limits cell-cycle progression. Cells that re-enter the cell cycle encounter replication stress that is well tolerated in physiologically sized cells but causes severe DNA damage in enlarged cells, ultimately resulting in mitotic failure and permanent cell-cycle withdrawal. We demonstrate that enlarged cells fail to recruit 53BP1 and other non-homologous end joining (NHEJ) machinery to DNA damage sites and fail to robustly initiate DNA damage-dependent p53 signaling, rendering them highly sensitive to genotoxic stress. We propose that an impaired DNA damage response primes enlarged cells for persistent replication-acquired damage, ultimately leading to cell division failure and permanent cell-cycle exit.
Topics: Cell Cycle; Cell Division; Cellular Senescence; Homeostasis; DNA Damage; Tumor Suppressor Protein p53
PubMed: 37977116
DOI: 10.1016/j.molcel.2023.10.018 -
Molecular Cell Jul 2023DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an...
DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.
Topics: Humans; RNA Polymerase II; DNA Replication; Transcription, Genetic; DNA Helicases; Genomic Instability
PubMed: 37295432
DOI: 10.1016/j.molcel.2023.05.015 -
Molecular Cell Aug 2023During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during...
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Replication; DNA Helicases; Saccharomyces cerevisiae; DNA, Single-Stranded
PubMed: 37506699
DOI: 10.1016/j.molcel.2023.06.035 -
Nature Jul 2023Immune checkpoint blockade (ICB) immunotherapy is a first-line treatment for selected cancers, yet the mechanisms of its efficacy remain incompletely understood.... (Review)
Review
Immune checkpoint blockade (ICB) immunotherapy is a first-line treatment for selected cancers, yet the mechanisms of its efficacy remain incompletely understood. Furthermore, only a minority of patients with cancer benefit from ICB, and there is a lack of fully informative treatment response biomarkers. Selectively exploiting defects in DNA damage repair is also a standard treatment for cancer, spurred by enhanced understanding of the DNA damage response (DDR). DDR and ICB are closely linked-faulty DDR produces immunogenic cancer neoantigens that can increase the efficacy of ICB therapy, and tumour mutational burden is a good but imperfect biomarker for the response to ICB. DDR studies in ICB efficacy initially focused on contributions to neoantigen burden. However, a growing body of evidence suggests that ICB efficacy is complicated by the immunogenic effects of nucleic acids generated from exogenous DNA damage or endogenous processes such as DNA replication. Chemotherapy, radiation, or selective DDR inhibitors (such as PARP inhibitors) can generate aberrant nucleic acids to induce tumour immunogenicity independently of neoantigens. Independent of their functions in immunity, targets of immunotherapy such as cyclic GMP-AMP synthase (cGAS) or PD-L1 can crosstalk with DDR or the DNA repair machinery to influence the response to DNA-damaging agents. Here we review the rapidly evolving, multifaceted interfaces between DDR, nucleic acid immunogenicity and immunotherapy efficacy, focusing on ICB. Understanding these interrelated processes could explain ICB treatment failures and reveal novel exploitable therapeutic vulnerabilities in cancers. We conclude by addressing major unanswered questions and new research directions.
Topics: Humans; Antigens, Neoplasm; DNA Damage; DNA Repair; Immunotherapy; Neoplasms; Nucleic Acids; DNA Replication; Immune Checkpoint Inhibitors; Mutation; Biomarkers, Tumor; Poly(ADP-ribose) Polymerase Inhibitors
PubMed: 37468584
DOI: 10.1038/s41586-023-06069-6