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Cell Chemical Biology Oct 2023Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer...
Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.
Topics: Humans; Proliferating Cell Nuclear Antigen; Chromatin; Protein Binding; Neoplasms; DNA; DNA Replication
PubMed: 37531956
DOI: 10.1016/j.chembiol.2023.07.001 -
Nature Reviews. Molecular Cell Biology Sep 2023Many cellular processes require large-scale rearrangements of chromatin structure. Structural maintenance of chromosomes (SMC) protein complexes are molecular machines... (Review)
Review
Many cellular processes require large-scale rearrangements of chromatin structure. Structural maintenance of chromosomes (SMC) protein complexes are molecular machines that can provide structure to chromatin. These complexes can connect DNA elements in cis, walk along DNA, build and processively enlarge DNA loops and connect DNA molecules in trans to hold together the sister chromatids. These DNA-shaping abilities place SMC complexes at the heart of many DNA-based processes, including chromosome segregation in mitosis, transcription control and DNA replication, repair and recombination. In this Review, we discuss the latest insights into how SMC complexes such as cohesin, condensin and the SMC5-SMC6 complex shape DNA to direct these fundamental chromosomal processes. We also consider how SMC complexes, by building chromatin loops, can counteract the natural tendency of alike chromatin regions to cluster. SMC complexes thus control nuclear organization by participating in a molecular tug of war that determines the architecture of our genome.
Topics: Chromosomes; Chromatin; DNA; DNA Replication; Mitosis; Cell Cycle Proteins
PubMed: 37231112
DOI: 10.1038/s41580-023-00609-8 -
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 -
Nature Cell Biology Dec 2023The intricate orchestration of enzymatic activities involving nicotinamide adenine dinucleotide (NAD) is essential for maintaining metabolic homeostasis and preserving...
The intricate orchestration of enzymatic activities involving nicotinamide adenine dinucleotide (NAD) is essential for maintaining metabolic homeostasis and preserving genomic integrity. As a co-enzyme, NAD plays a key role in regulating metabolic pathways, such as glycolysis and Kreb's cycle. ADP-ribosyltransferases (PARPs) and sirtuins rely on NAD to mediate post-translational modifications of target proteins. The activation of PARP1 in response to DNA breaks leads to rapid depletion of cellular NAD compromising cell viability. Therefore, the levels of NAD must be tightly regulated. Here we show that exogenous NAD, but not its precursors, has a direct effect on mitochondrial activity. Short-term incubation with NAD boosts Kreb's cycle and the electron transport chain and enhances pyrimidine biosynthesis. Extended incubation with NAD results in depletion of pyrimidines, accumulation of purines, activation of the replication stress response and cell cycle arrest. Moreover, a combination of NAD and 5-fluorouridine selectively kills cancer cells that rely on de novo pyrimidine synthesis. We propose an integrated model of how NAD regulates nucleotide metabolism, with relevance to healthspan, ageing and cancer therapy.
Topics: NAD; Glycolysis; Metabolic Networks and Pathways; Genomics; DNA Replication
PubMed: 37957325
DOI: 10.1038/s41556-023-01280-z -
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 -
Nature Chemical Biology Dec 2023Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can...
Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in Bacillus thuringiensis with an engineered error-prone orthogonal DNA polymerase. BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells.
Topics: DNA, Bacterial; DNA-Directed DNA Polymerase; Plasmids; Saccharomyces cerevisiae; DNA Replication; Bacteria; Bacillus subtilis
PubMed: 37443393
DOI: 10.1038/s41589-023-01387-2 -
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 -
Molecular Cell Oct 2023Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal...
Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.
Topics: Humans; Cell Survival; Chromatin; Cyclin E; DNA Damage; DNA Repair; DNA Replication; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Neoplasms
PubMed: 37591242
DOI: 10.1016/j.molcel.2023.07.026