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The EMBO Journal Jan 2023EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and...
EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA-DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.
Topics: RNA; Exoribonucleases; Kinetics; DNA; Exodeoxyribonucleases
PubMed: 36326837
DOI: 10.15252/embj.2022111703 -
DNA Repair Aug 2015DNA mismatch repair (MMR) acts to repair mispaired bases resulting from misincorporation errors during DNA replication and also recognizes mispaired bases in... (Review)
Review
DNA mismatch repair (MMR) acts to repair mispaired bases resulting from misincorporation errors during DNA replication and also recognizes mispaired bases in recombination (HR) intermediates. Exonuclease 1 (Exo1) is a 5' → 3' exonuclease that participates in a number of DNA repair pathways. Exo1 was identified as an exonuclease that participates in Saccharomyces cerevisiae and human MMR where it functions to excise the daughter strand after mispair recognition, and additionally Exo1 functions in end resection during HR. However, Exo1 is not absolutely required for end resection during HR in vivo. Similarly, while Exo1 is required in MMR reactions that have been reconstituted in vitro, genetics studies have shown that it is not absolutely required for MMR in vivo suggesting the existence of Exo1-independent and Exo1-dependent MMR subpathways. Here, we review what is known about the Exo1-independent and Exo1-dependent subpathways, including studies of mutations in MMR genes that specifically disrupt either subpathway.
Topics: Base Pair Mismatch; DNA Breaks, Double-Stranded; DNA Mismatch Repair; DNA Replication; DNA, Fungal; Exodeoxyribonucleases; Gene Expression Regulation, Fungal; Humans; Models, Molecular; Mutation; Protein Structure, Secondary; Protein Structure, Tertiary; Saccharomyces cerevisiae
PubMed: 25956862
DOI: 10.1016/j.dnarep.2015.04.010 -
Methods in Enzymology 2019Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic...
Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic acid sensing and immune activation in humans and mice. Mutations in the TREX1 gene cause a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome, familial chilblain lupus, retinal vasculopathy with cerebral leukodystrophy, and are associated with systemic lupus erythematosus. More than 60 disease-causing TREX1 variants have been identified including dominant and recessive, missense, and frameshift mutations that map to the catalytic core region and to the C-terminal cell localization region. The TREX1-disease causing mutations affect exonuclease activity at varied levels. In this chapter, we describe methods to purify variant recombinant TREX1 enzymes and measure the exonuclease activity using ssDNA and dsDNA substrates. The relationships between TREX1 activities, types of TREX1 mutations, and TREX1-associated autoimmune diseases are considered.
Topics: Animals; Autoimmunity; Exodeoxyribonucleases; Humans; Phosphoproteins
PubMed: 31455522
DOI: 10.1016/bs.mie.2019.05.004 -
DNA Repair Mar 2011Homology-dependent repair of DNA double-strand breaks (DSBs) initiates by the 5'-3' resection of the DNA ends to create single-stranded DNA (ssDNA), the substrate for... (Review)
Review
Homology-dependent repair of DNA double-strand breaks (DSBs) initiates by the 5'-3' resection of the DNA ends to create single-stranded DNA (ssDNA), the substrate for Rad51/RecA binding. Long tracts of ssDNA are also required for activation of the ATR-mediated checkpoint response. Thus, identifying the proteins required and the underlying mechanism for DNA end resection has been an intense area of investigation. Genetic studies in Saccharomyces cerevisiae show that end resection takes place in two steps. Initially, a short oligonucleotide tract is removed from the 5' strand to create an early intermediate with a short 3' overhang. Then in a second step the early intermediate is rapidly processed generating an extensive tract of ssDNA. The first step is dependent on the highly conserved Mre11-Rad50-Xrs2 complex and Sae2, while the second step employs the exonuclease Exo1 and/or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 with the endonuclease Dna2. Here we review recent in vitro and in vivo findings that shed more light into the mechanisms of DSB processing in mitotic and meiotic DSB repair as well as in telomere metabolism.
Topics: Animals; DNA; DNA Breaks, Double-Stranded; Exodeoxyribonucleases; Humans; RecQ Helicases
PubMed: 21227759
DOI: 10.1016/j.dnarep.2010.12.004 -
G3 (Bethesda, Md.) Jan 2021Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex not only allows bacteria to repair DNA double-strand breaks but...
Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex not only allows bacteria to repair DNA double-strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. In this study, we detected putative Chi motifs in a large dataset of Proteobacteria and identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.
Topics: DNA, Bacterial; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Proteobacteria; Recombination, Genetic
PubMed: 33561247
DOI: 10.1093/g3journal/jkaa054 -
Journal of the American Chemical Society Jan 2021The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is...
The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.
Topics: Aptamers, Nucleotide; Digestion; Escherichia coli; Exodeoxyribonucleases; SELEX Aptamer Technique
PubMed: 33378616
DOI: 10.1021/jacs.0c09559 -
Microbiological Reviews Mar 1988
Review
Topics: Diploidy; Exodeoxyribonuclease V; Exodeoxyribonucleases; Gene Expression Regulation; Genes, Bacterial; Rec A Recombinases; Recombination, Genetic
PubMed: 3280962
DOI: 10.1128/mr.52.1.1-28.1988 -
Microbiological Reviews Sep 1994Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products... (Review)
Review
Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
Topics: Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Models, Genetic; Rec A Recombinases; Recombination, Genetic
PubMed: 7968921
DOI: 10.1128/mr.58.3.401-465.1994 -
Scientific Reports Jan 2016DNA repair mechanisms are responsible for maintaining the integrity of DNA and are essential to life. However, our knowledge of DNA repair mechanisms is based on model...
DNA repair mechanisms are responsible for maintaining the integrity of DNA and are essential to life. However, our knowledge of DNA repair mechanisms is based on model organisms such as Escherichia coli, and little is known about free living and uncultured microorganisms. In this study, a functional screening was applied in a metagenomic library with the goal of discovering new genes involved in the maintenance of genomic integrity. One clone was identified and the sequence analysis showed an open reading frame homolog to a hypothetical protein annotated as a member of the Exo_Endo_Phos superfamily. This novel enzyme shows 3'-5' exonuclease activity on single and double strand DNA substrates and it is divalent metal-dependent, EDTA-sensitive and salt resistant. The clone carrying the hypothetical ORF was able to complement strains deficient in recombination or base excision repair, suggesting that the new enzyme may be acting on the repair of single strand breaks with 3' blockers, which are substrates for these repair pathways. Because this is the first report of an enzyme obtained from a metagenomic approach showing exonuclease activity, it was named ExoMeg1. The metagenomic approach has proved to be a useful tool for identifying new genes of uncultured microorganisms.
Topics: Exodeoxyribonucleases; Genomic Library; Metagenome
PubMed: 26815639
DOI: 10.1038/srep19712 -
Cell Nov 1999
Review
Topics: Cysteine Endopeptidases; Exodeoxyribonuclease V; Exodeoxyribonucleases; Humans; Multienzyme Complexes; Proteasome Endopeptidase Complex; RNA
PubMed: 10571176
DOI: 10.1016/s0092-8674(00)81520-2