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Replication fork

Xiubao Chang, Yuexian Hou
Genome editing is a powerful tool to modify a specific gene and to correct a disease-causing mutation. Recently developed new techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9), significantly facilitate the progression in this field. However, mutations associated with the double strand DNA breaks (DSBs) introduced by these systems hampered their direct usage in clinic. In order to prevent the mutations caused by DSBs, we have designed a novel mean to induce homology-directed recombination (HDR) without DSBs, i...
2018: International Journal of Biochemistry and Molecular Biology
Élodie Chrabaszcz, Luisa Laureti, Vincent Pagès
The genome of all organisms is constantly threatened by numerous agents that cause DNA damage. When the replication fork encounters an unrepaired DNA lesion, two DNA damage tolerance pathways are possible: error-prone translesion synthesis (TLS) that requires specialized DNA polymerases, and error-free damage avoidance that relies on homologous recombination (HR). The balance between these two mechanisms is essential since it defines the level of mutagenesis during lesion bypass, allowing genetic variability and adaptation to the environment, but also introduces the risk of generating genome instability...
February 26, 2018: Nucleic Acids Research
Anurag Kumar Sinha, Christophe Possoz, Adeline Durand, Jean-Michel Desfontaines, François-Xavier Barre, David R F Leach, Bénédicte Michel
It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon...
March 9, 2018: PLoS Genetics
Clara B García-Calderón, José Antonio Bejarano-García, Isabel Tinoco-Gago, María José Castro, Paula Moreno-Gordillo, José I Piruat, Teresa Caballero-Velázquez, José A Pérez-Simón, Iván V Rosado
Metabolically reactive formaldehyde is a genotoxin and a carcinogen. Mice lacking the main formaldehyde-detoxifying gene Adh5 combined with the loss of the Fanconi anemia (FA) DNA repair pathway rapidly succumbed to bone marrow failure (BMF) primarily due to the extensive ablation of the hematopoietic stem cell (HSC) pool. However, the mechanism by which formaldehyde mediates these toxic effects is still unknown. We uncover a detrimental role of tetrahydrofolic acid (THF) in cells lacking Adh5 or the FA repair pathway...
March 6, 2018: Cell Death and Differentiation
Valerie S Forsyth, Chelsie E Armbruster, Sara N Smith, Ali Pirani, A Cody Springman, Matthew S Walters, Greta R Nielubowicz, Stephanie D Himpsl, Evan S Snitkin, Harry L T Mobley
Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division...
March 6, 2018: MBio
Mary-Katharine Collins, Kaitlin Peters, Joseph C English, Peter Rady, Stephen Tyring, Jaroslaw Jedrych
Schimke immune-osseous dysplasia1 (SIOD) is an autosomal recessive disorder caused by mutations in the SMARCAL1 gene encoding an ATP-driven annealing helicase that stabilizes stalled replication forks and facilitates DNA repair during replication2 . The syndrome is characterized by spondyloepiphyseal dysplasia, steroid-resistant nephropathy and cellular immune deficiency.
March 2, 2018: Journal of Cutaneous Pathology
Dana Branzei, Michele Giannattasio
Roy et al. (2018. J. Cell. Biol. describe an ingenious single-cell assay system, in situ analysis of protein interactions at DNA replication forks (SIRF), for the quantitative analysis of protein interactions with nascent DNA at active and stalled replication forks. The sensitive and accurate SIRF methodology is suitable for multiparameter measurements in cell populations.
March 1, 2018: Journal of Cell Biology
Debadrita Bhattacharya, Disha Hiregange, Basuthkar J Rao
In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogation leads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition of ATR kinase activity specifically during replication stress recovery results in persistent ATR signalling, evidenced by the presence of ATR-dependent phosphorylation marks (gamma H2AX, pChk1 and pRad17) and delayed cell cycle re-entry. Further, such disruption of ATR signalling attenuation leads to double-strand breaks, fork collapse and thereby 'replication catastrophe'...
March 2018: Journal of Biosciences
Barbara Bukowska, Boleslaw T Karwowski
The clustered DNA lesions are a characteristic feature of ionizing radiation and are defined as two or more damage sites formed within 20 bps after the passage of a single radiation track. The clustered DNA lesions are divided into two major groups: double-stranded breaks (DSBs) and non-DSB clusters also known as Oxidatively-induced Clustered DNA Lesions (OCDLs), which could involve either two opposing strands or the same strand. As irradiation is gaining greater interest in cancer treatment as well as in imaging techniques, the detailed knowledge of its genotoxicity and the mechanisms of repair of radiation-induced DNA damage remain issues to explore...
February 25, 2018: Current Medicinal Chemistry
F Berardinelli, A Sgura, A Facoetti, S Leone, B Vischioni, M Ciocca, A Antoccia
The pentacyclic acridine RHPS4 is a highly potent and specific G-quadruplex (G4) ligand, which binds and stabilizes telomeric G4 leading to the block of the replication forks at telomeres and consequently to telomere dysfunctionalization. In turn, the cell recognizes unprotected telomeres as DNA double-strand breaks with consequent activation of DNA repair response at telomeres, cellular growth impairment and death. Data from the literature showed the capability of this compound to sensitize U251MG glioblastoma radioresistant cell line to X-rays sparsely ionizing radiations...
February 27, 2018: FEBS Journal
Mário Špírek, Jarmila Mlcoušková, Ondrej Belán, Máté Gyimesi, Gábor M Harami, Eszter Molnár, Jiri Novacek, Mihály Kovács, Lumir Krejci
Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an 'open' conformation when compared to a 'closed' structure formed by mutants, reflecting alterations in helical pitch...
February 22, 2018: Nucleic Acids Research
Sunetra Roy, Jessica W Luzwick, Katharina Schlacher
DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding...
February 23, 2018: Journal of Cell Biology
Jessica Fletcher, Liam Griffiths, Thomas Caspari
The S. pombe checkpoint kinase, Cds1, protects the integrity of stalled DNA replication forks after its phosphorylation at threonine-11 by Rad3 (ATR). Modified Cds1 associates through its N-terminal forkhead-associated domain (FHA)-domain with Mrc1 (Claspin) at stalled forks. We report here that nutrient starvation results in post-translational changes to Cds1 and the loss of Mrc1. A drop in glucose after a down-shift from 3% to 0.1-0.3%, or when cells enter the stationary phase, triggers a sharp decline in Mrc1 and the accumulation of insoluble Cds1...
February 23, 2018: Cells
Sharon B Cantor, Jennifer A Calvo
The BRCA-Fanconi anemia (FA) pathway preserves the genome and suppresses cancer and is a main determinant of chemotherapeutic efficacy. The hereditary breast cancer genes BRCA1 and BRCA2 function in DNA double-strand break repair mediating distinct steps of homologous recombination (HR). More recently, independent of DNA repair, functions in the replication stress response have come to light, providing insight as to how the BRCA-FA pathway also balances genome preservation with proliferation. The BRCA-FA proteins associate with the replisome and contribute to the efficiency and recovery of replication following perturbations that slow or arrest DNA replication...
February 22, 2018: Cold Spring Harbor Symposia on Quantitative Biology
George E Ronson, Ann Liza Piberger, Martin R Higgs, Anna L Olsen, Grant S Stewart, Peter J McHugh, Eva Petermann, Nicholas D Lakin
PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection...
February 21, 2018: Nature Communications
Ray Kreienkamp, Simona Graziano, Nuria Coll-Bonfill, Gonzalo Bedia-Diaz, Emily Cybulla, Alessandro Vindigni, Dale Dorsett, Nard Kubben, Luis Francisco Zirnberger Batista, Susana Gonzalo
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease caused by a truncated lamin A protein (progerin) that drives cellular and organismal decline. HGPS patient-derived fibroblasts accumulate genomic instability, but its underlying mechanisms and contribution to disease remain poorly understood. Here, we show that progerin-induced replication stress (RS) drives genomic instability by eliciting replication fork (RF) stalling and nuclease-mediated degradation. Rampant RS is accompanied by upregulation of the cGAS/STING cytosolic DNA sensing pathway and activation of a robust STAT1-regulated interferon (IFN)-like response...
February 20, 2018: Cell Reports
Morgane Macheret, Thanos D Halazonetis
Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes...
February 21, 2018: Nature
Ying Wai Chan, Stephen C West
Successful chromosome segregation depends on the timely removal of DNA recombination and replication intermediates that interlink sister chromatids. These intermediates are acted upon by structure-selective endonucleases that promote incisions close to the junction point. GEN1, a member of the Rad2/XPG endonuclease family, was identified on the basis of its ability to cleave Holliday junction recombination intermediates. Resolution occurs by a nick and counter-nick mechanism in strands that are symmetrically related across the junction point, leading to the formation of ligatable nicked duplex products...
2018: Methods in Enzymology
Catharina von Nicolai, Åsa Ehlén, Juan S Martinez, Aura Carreira
Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break...
2018: Methods in Enzymology
Shyamal Subramanyam, Colin D Kinz-Thompson, Ruben L Gonzalez, Maria Spies
Human RAD51 promotes accurate DNA repair by homologous recombination and is involved in protection and repair of damaged DNA replication forks. The active species of RAD51 and related recombinases in all organisms is a nucleoprotein filament assembled on single-stranded DNA (ssDNA). The formation of a nucleoprotein filament competent for the recombination reaction, or for DNA replication support, is a delicate and strictly regulated process, which occurs through filament nucleation followed by filament extension...
2018: Methods in Enzymology
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