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Nucleic Acids Research Nov 2022Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside...
Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.
Topics: Bacterial Proteins; Exonucleases; Nucleotides; RNA; Firmicutes
PubMed: 36478094
DOI: 10.1093/nar/gkac1091 -
Bioinformatics (Oxford, England) Jul 2021High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is...
MOTIVATION
High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is a modified protocol, which uses lambda exonuclease to digest DNA close to the TF-DNA complex, in order to improve on the positional resolution of the TF-DNA contact. Because the digestion occurs in the 5'-3' orientation, the protocol produces directional footprints close to the complex, on both sides of the double stranded DNA. Like all ChIP-based methods, ChIP-exo reports a mixture of different regions associated with the TF: those bound directly to the TF as well as via intermediaries. However, the distribution of footprints are likely to be indicative of the complex forming at the DNA.
RESULTS
We present ExoDiversity, which uses a model-based framework to learn a joint distribution over footprints and motifs, thus resolving the mixture of ChIP-exo footprints into diverse binding modes. It uses no prior motif or TF information and automatically learns the number of different modes from the data. We show its application on a wide range of TFs and organisms/cell-types. Because its goal is to explain the complete set of reported regions, it is able to identify co-factor TF motifs that appear in a small fraction of the dataset. Further, ExoDiversity discovers small nucleotide variations within and outside canonical motifs, which co-occur with variations in footprints, suggesting that the TF-DNA structural configuration at those regions is likely to be different. Finally, we show that detected modes have specific DNA shape features and conservation signals, giving insights into the structure and function of the putative TF-DNA complexes.
AVAILABILITY AND IMPLEMENTATION
The code for ExoDiversity is available on https://github.com/NarlikarLab/exoDIVERSITY.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Binding Sites; Chromatin Immunoprecipitation; DNA; DNA Footprinting; Exonucleases; Protein Binding; Sequence Analysis, DNA
PubMed: 34252930
DOI: 10.1093/bioinformatics/btab274 -
Proceedings of the National Academy of... Mar 2018Inorganic P is involved in all major biochemical pathways. Here we describe a previously unreported activity of P We show that P and its structural mimics, vanadate and...
Inorganic P is involved in all major biochemical pathways. Here we describe a previously unreported activity of P We show that P and its structural mimics, vanadate and arsenate, enhance nascent transcript cleavage by RNA polymerase (RNAP). They engage an Mg ion in catalysis and activate an attacking water molecule. P, vanadate, and arsenate stimulate the intrinsic exonuclease activity of the enzyme nearly 2,000-fold at saturating concentrations of the reactant anions and Mg This enhancement is comparable to that of specialized transcript cleavage protein factors Gre and TFIIS (3,000- to 4,000-fold). Unlike these protein factors, P and its analogs do not stimulate endonuclease transcript cleavage. Conversely, the protein factors only marginally enhance exonucleolytic cleavage. P thus complements cellular protein factors in assisting hydrolytic RNA cleavage by extending the repertoire of RNAP transcript degradation modes.
Topics: Arsenates; Biocatalysis; DNA-Directed RNA Polymerases; Exonucleases; Hydrolysis; Kinetics; Phosphates; RNA; Transcription, Genetic; Transcriptional Elongation Factors; Vanadates
PubMed: 29483274
DOI: 10.1073/pnas.1720370115 -
Nature Communications Feb 2023The degradation process of RNA is decisive in guaranteeing high-fidelity translation of genetic information in living organisms. However, visualizing the single-base...
The degradation process of RNA is decisive in guaranteeing high-fidelity translation of genetic information in living organisms. However, visualizing the single-base degradation process in real time and deciphering the degradation mechanism at the single-enzyme level remain formidable challenges. Here, we present a reliable in-situ single-PNPase-molecule dynamic electrical detector based on silicon nanowire field-effect transistors with ultra-high temporal resolution. These devices are capable of realizing real-time and label-free monitoring of RNA analog degradation with single-base resolution, including RNA analog binding, single-nucleotide hydrolysis, and single-base movement. We discover a binding event of the enzyme (near the active site) with the nucleoside, offering a further understanding of the RNA degradation mechanism. Relying on systematic analyses of independent reads, approximately 80% accuracy in RNA nucleoside sequencing is achieved in a single testing process. This proof-of-concept sets up a Complementary Metal Oxide Semiconductor (CMOS)-compatible playground for the development of high-throughput detection technologies toward mechanistic exploration and single-molecule sequencing.
Topics: Exonucleases; Nucleosides; RNA; Sequence Analysis, RNA; RNA Stability
PubMed: 36725855
DOI: 10.1038/s41467-023-36278-6 -
RNA (New York, N.Y.) May 2022The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act...
The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical GpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5' monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5' exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5' monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5' end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.
Topics: Exonucleases; Exoribonucleases; Nuclear Proteins; RNA; RNA, Ribosomal; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcriptome
PubMed: 35140172
DOI: 10.1261/rna.078952.121 -
Nature Communications Oct 2018Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a...
Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while Mg and Mg have similar binding constants, the dissociation rate of Mg is two order of magnitude lower than that of Mg due to a kinetic-barrier-difference. At physiological Mg concentration, the Mg ion near the 5'-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of Mg result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.
Topics: Biocatalysis; Calcium; DNA; Exonucleases; Fluorescence; Fluorescence Resonance Energy Transfer; Ions; Kinetics; Magnesium; Metals; Models, Molecular; Single Molecule Imaging
PubMed: 30353000
DOI: 10.1038/s41467-018-06750-9 -
Genome Research Nov 2014Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated...
Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.
Topics: Ataxia Telangiectasia Mutated Proteins; Class Ia Phosphatidylinositol 3-Kinase; Codon, Nonsense; DNA Mutational Analysis; DNA Polymerase II; DNA Polymerase III; DNA Replication; Databases, Genetic; Exonucleases; Genome-Wide Association Study; Humans; Microsatellite Instability; Models, Molecular; Mutation, Missense; Neoplasms; Phosphatidylinositol 3-Kinases; Protein Structure, Tertiary; Replication Origin; Tumor Suppressor Protein p53
PubMed: 25228659
DOI: 10.1101/gr.174789.114 -
Nature Communications Oct 2017High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1...
High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.
Topics: Cryoelectron Microscopy; DNA Replication; Deoxyribonuclease IV (Phage T4-Induced); Exodeoxyribonucleases; Molecular Structure; Mycobacterium tuberculosis; Zinc
PubMed: 29021523
DOI: 10.1038/s41467-017-00886-w -
Genes & Development Feb 2023Pol2 is the leading-strand DNA polymerase in budding yeast. Here we describe an antagonism between its conserved POPS (Pol2 family-specific catalytic core peripheral...
Pol2 is the leading-strand DNA polymerase in budding yeast. Here we describe an antagonism between its conserved POPS (Pol2 family-specific catalytic core peripheral subdomain) and exonuclease domain and the importance of this antagonism in genome replication. We show that multiple defects caused by POPS mutations, including impaired growth and DNA synthesis, genome instability, and reliance on other genome maintenance factors, were rescued by exonuclease inactivation. Single-molecule data revealed that the rescue stemmed from allowing sister replication forks to progress at equal rates. Our data suggest that balanced activity of Pol2's POPS and exonuclease domains is vital for genome replication and stability.
Topics: Humans; Exonucleases; DNA Replication; Mutation; Genomic Instability; DNA Polymerase II
PubMed: 36702483
DOI: 10.1101/gad.350054.122 -
Genetics Jun 2014Members of the RecQ family of helicases are known for their roles in DNA repair, replication, and recombination. Mutations in the human RecQ helicases, WRN and BLM,...
Members of the RecQ family of helicases are known for their roles in DNA repair, replication, and recombination. Mutations in the human RecQ helicases, WRN and BLM, cause Werner and Bloom syndromes, which are diseases characterized by genome instability and an increased risk of cancer. While WRN contains both a helicase and an exonuclease domain, the Drosophila melanogaster homolog, WRNexo, contains only the exonuclease domain. Therefore the Drosophila model system provides a unique opportunity to study the exonuclease functions of WRN separate from the helicase. We created a null allele of WRNexo via imprecise P-element excision. The null WRNexo mutants are not sensitive to double-strand break-inducing reagents, suggesting that the exonuclease does not play a key role in homologous recombination-mediated repair of DSBs. However, WRNexo mutant embryos have a reduced hatching frequency and larvae are sensitive to the replication fork-stalling reagent, hydroxyurea (HU), suggesting that WRNexo is important in responding to replication stress. The role of WRNexo in the HU-induced stress response is independent of Rad51. Interestingly, the hatching defect and HU sensitivity of WRNexo mutants do not occur in flies containing an exonuclease-dead copy of WRNexo, suggesting that the role of WRNexo in replication is independent of exonuclease activity. Additionally, WRNexo and Blm mutants exhibit similar sensitivity to HU and synthetic lethality in combination with mutations in structure-selective endonucleases. We propose that WRNexo and BLM interact to promote fork reversal following replication fork stalling and in their absence regressed forks are restarted through a Rad51-mediated process.
Topics: Animals; DNA Breaks, Double-Stranded; DNA Helicases; DNA Repair; DNA Replication; Drosophila Proteins; Drosophila melanogaster; Exonucleases; Female; Gene Expression Regulation, Developmental; Hydroxyurea; Male; Mutation; Rad51 Recombinase; RecQ Helicases
PubMed: 24709634
DOI: 10.1534/genetics.114.164228