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Science (New York, N.Y.) Aug 2023Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas...
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR-Cas9-based synthetic lethal screens in cancer cells, we identified subunits of the 9-1-1 complex (RAD9A-RAD1-HUS1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncovered an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes Polo-like kinase 1 (PLK1) phosphorylation, and interacts with polymerase θ (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs that originate in S phase. Our findings offer insights into the synthetic lethal relationship between the genes and and and the synergistic effect of Polθ and poly(ADP-ribose) polymerase (PARP) inhibitors.
Topics: Humans; Cell Cycle Proteins; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Exonucleases; HEK293 Cells; Mitosis; Poly(ADP-ribose) Polymerases
PubMed: 37440612
DOI: 10.1126/science.adh3694 -
Nature Jun 2023CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for...
CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; Cryoelectron Microscopy; DNA; Exonucleases; Integrases; Biocatalysis; Genome, Bacterial
PubMed: 37316664
DOI: 10.1038/s41586-023-06178-2 -
DNA Repair Sep 2022The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by... (Review)
Review
The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by the three major replicases, DNA polymerases α, δ and ε. When an incorrect base is occasionally inserted, the latter Pols δ and ε also have a 3 ´ to 5 ´ exonuclease activity that can remove the mismatch to allow correct DNA synthesis to proceed. Lastly, rare mismatches that escape proofreading activity and are present in newly replicated DNA can be removed by DNA mismatch repair. In this review, we consider evidence supporting the hypothesis that the second mechanism, proofreading, can operate in two different ways. Primer terminal mismatches made by either Pol δ or Pol ε can be 'intrinsically' proofread. This mechanism occurs by direct transfer of a misinserted base made at the polymerase active site to the exonuclease active site that is located a short distance away. Intrinsic proofreading allows mismatch excision without intervening enzyme dissociation. Alternatively, considerable evidence suggests that mismatches made by any of the three replicases can also be proofread by 'extrinsic' proofreading by Pol δ. Extrinsic proofreading occurs when a mismatch made by any of the three replicases is initially abandoned, thereby allowing the exonuclease active site of Pol δ to bind directly to and remove the mismatch before replication continues. Here we review the evidence that extrinsic proofreading significantly enhances the fidelity of nuclear DNA replication, and we then briefly consider the implications of this process for evolution and disease.
Topics: DNA; DNA Polymerase II; DNA Polymerase III; DNA Replication; Exonucleases
PubMed: 35850061
DOI: 10.1016/j.dnarep.2022.103369 -
Nature Communications Dec 2023DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously...
DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.
Topics: Humans; DNA Replication; DNA-Directed DNA Polymerase; DNA; Exonucleases
PubMed: 38151585
DOI: 10.1038/s41467-023-44198-8 -
Genes Aug 2022Cardiomyopathies comprise a heterogeneous group of cardiac diseases identified by myocardium disorders and diminished cardiac function. They often lead to heart failure... (Review)
Review
Cardiomyopathies comprise a heterogeneous group of cardiac diseases identified by myocardium disorders and diminished cardiac function. They often lead to heart failure or heart transplantation and constitute one of the principal causes of morbidity and mortality worldwide. Circular RNAs (circRNAs) are a novel type of noncoding RNAs. They are covalently closed and single-stranded and derived from the exons and introns of genes by alternative splicing. This specific structure renders them resistant to exonuclease digestion. Many recent studies have demonstrated that circRNAs are highly abundant and conserved and can play central roles in biological functions such as microRNA (miRNA) sponging, splicing, and transcription regulation. Emerging evidence indicates that circRNAs can play significant roles in cardiovascular diseases, including cardiomyopathies. In this review, we briefly describe the current understanding regarding the classification, nomenclature, characteristics, and function of circRNAs and report recent significant findings concerning the roles of circRNAs in cardiomyopathies. Furthermore, we discuss the clinical application potential of circRNAs as the therapeutic targets and diagnostic biomarkers of cardiomyopathies.
Topics: Alternative Splicing; Biomarkers; Cardiomyopathies; Exonucleases; Humans; MicroRNAs; RNA, Circular
PubMed: 36140705
DOI: 10.3390/genes13091537 -
Current Genetics Oct 2018DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative... (Review)
Review
DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative DNA polymerases is to replicate DNA with the very high accuracy. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Proofreading activity that assists most of the replicative polymerases is responsible for removal of incorrectly incorporated nucleotides from the primer terminus before further primer extension. It is estimated that proofreading improves the fidelity by a 2-3 orders of magnitude. The primer with the incorrect terminal nucleotide has to be moved to exonuclease active site, and after removal of the wrong nucleotide must be transferred back to polymerase active site. The mechanism that allows the transfer of the primer between pol and exo site is not well understood. While defects in MMR are well known to be linked with increased cancer incidence only recently, the replicative polymerases that have alterations in the exonuclease domain have been associated with some sporadic and hereditary human cancers. In this review, we would like to emphasize the importance of proofreading (3'-5' exonuclease activity) in the fidelity of DNA replication and to highlight what is known about switching from polymerase to exonuclease active site.
Topics: Catalytic Domain; DNA Mismatch Repair; DNA Replication; DNA-Directed DNA Polymerase; Exonucleases; Humans; Mutation; Protein Conformation
PubMed: 29500597
DOI: 10.1007/s00294-018-0820-1 -
Nature Reviews. Microbiology Nov 2008Bacteriophages are central components in the development of molecular tools for microbial genetics. Mycobacteriophages have proven to be a rich resource for tuberculosis... (Review)
Review
Bacteriophages are central components in the development of molecular tools for microbial genetics. Mycobacteriophages have proven to be a rich resource for tuberculosis genetics, and the recent development of a mycobacterial recombineering system based on mycobacteriophage Che9c-encoded proteins offers new approaches to mycobacterial mutagenesis. Expression of the phage exonuclease and recombinase substantially enhances recombination frequencies in both fast- and slow-growing mycobacteria, thereby facilitating construction of both gene knockout and point mutants; it also provides a simple and efficient method for constructing mycobacteriophage mutants. Exploitation of host-specific phages thus provides a general strategy for recombineering and mutagenesis in genetically naive systems.
Topics: Exonucleases; Gene Deletion; Mutagenesis; Mycobacteriophages; Mycobacterium; Point Mutation; Recombinases; Viral Proteins
PubMed: 18923412
DOI: 10.1038/nrmicro2014 -
Methods in Enzymology 2022Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and...
Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and helicase, catalyze continuous DNA synthesis on the leading strand of the parental DNA duplex while the lagging strand is synthesized discontinuously. The helicase and DNA polymerase on their own are catalytically inefficient and weak motors for unwinding/replicating double-stranded DNA. However, when a helicase and DNA polymerase are functionally and physically coupled, they catalyze fast and highly processive leading strand DNA synthesis. DNA polymerase has a 3'-5' exonuclease activity, which removes nucleotides misincorporated in the nascent DNA. DNA synthesis kinetics, processivity, and accuracy are governed by the interplay of the helicase, DNA polymerase, and exonuclease activities within the replisome. This chapter describes quantitative biochemical and biophysical methods to study the coupling of these three critical activities during DNA replication. The methods include real-time quantitation of kinetics of DNA unwinding-synthesis by a coupled helicase-DNA polymerase complex, a 2-aminopurine fluorescence-based assay to map the precise positions of helicase and DNA polymerase with respect to the replication fork junction, and a radiometric assay to study the coupling of DNA polymerase, exonuclease, and helicase activities during processive leading strand DNA synthesis. These methods are presented here with bacteriophage T7 replication proteins as an example but can be applied to other systems with appropriate modifications.
Topics: DNA; DNA Helicases; DNA Replication; DNA-Directed DNA Polymerase; Exonucleases
PubMed: 35934486
DOI: 10.1016/bs.mie.2022.03.011 -
SLAS Discovery : Advancing Life... Jul 2021Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.
Topics: Antiviral Agents; COVID-19; Cloning, Molecular; Enzyme Assays; Enzyme Inhibitors; Escherichia coli; Exonucleases; Exoribonucleases; Gene Expression; Genetic Vectors; High-Throughput Screening Assays; Humans; Kinetics; RNA, Viral; Recombinant Proteins; SARS-CoV-2; Small Molecule Libraries; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Viral Nonstructural Proteins; Virus Replication
PubMed: 33870746
DOI: 10.1177/24725552211008854 -
Virologica Sinica Aug 2022ISG20 is an interferon-inducible exonuclease that inhibits virus replication. Although ISG20 is thought to degrade viral RNA, the antiviral mechanism and specificity of...
ISG20 is an interferon-inducible exonuclease that inhibits virus replication. Although ISG20 is thought to degrade viral RNA, the antiviral mechanism and specificity of ISG20 remain unclear. In this study, the antiviral role of ovine ISG20 (oISG20) in bluetongue virus (BTV) infection was investigated. It was found that BTV infection up-regulated the transcription of ovine ISG20 (oISG20) in a time- and BTV multiplicity of infection (MOI)-dependent manner. Overexpression of oISG20 suppressed the production of BTV genome, proteins, and virus titer, whereas the knockdown of oISG20 increased viral replication. oISG20 was found to co-localize with BTV proteins VP4, VP5, VP6, and NS2, but only directly interacted with VP4. Exonuclease defective oISG20 significantly decreased the inhibitory effect on BTV replication. In addition, the interaction of mutant oISG20 and VP4 was weakened, suggesting that binding to VP4 was associated with the inhibition of BTV replication. The present data characterized the anti-BTV effect of oISG20, and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.
Topics: Animals; Antiviral Agents; Bluetongue; Bluetongue virus; Exonucleases; Sheep; Virus Replication
PubMed: 35513266
DOI: 10.1016/j.virs.2022.04.010