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NAR Top Articles - Nucleic Acid Enzymes

Nucleic Acid Enzymes

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June 2015

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
Fonfara, I; Le Rhun, A; Chylinski, K; Makarova, KS; Lecrivain, AL; Bzdrenga, J; Koonin, EV; Charpentier, E
Nucleic Acids Res. 2014, 42, 2577-2590
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The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci...

Binary recombinase systems for high-resolution conditional mutagenesis
Hermann, M; Stillhard, P; Wildner, H; Seruggia, D; Kapp, V; Sanchez-Iranzo, H; Mercader, N; Montoliu, L; Zeilhofer, HU; Pelczar, P
Nucleic Acids Res. 2014, 42, 3894-3907
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Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering
Boissel, S; Jarjour, J; Astrakhan, A; Adey, A; Gouble, A; Duchateau, P; Shendure, J; Stoddard, BL; Certo, MT; Baker, D; Scharenberg, AM
Nucleic Acids Res. 2014, 42, 2591-2601
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Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

eIF4B, eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle
Harms, U; Andreou, AZ; Gubaev, A; Klostermeier, D
Nucleic Acids Res. 2014, 42, 7911-7922
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Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5 '-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions...

Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT
Xi, LH; Cech, TR
Nucleic Acids Res. 2014, 42, 8565-8577
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Telomerase is the ribonucleoprotein (RNP) enzyme that elongates telomeric DNA to compensate for the attrition occurring during each cycle of DNA replication. Knowing the levels of telomerase in continuously dividing cells is important for understanding how much telomerase is required for cell immortality. In this study, we measured the endogenous levels of the human telomerase RNP and its two key components, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). We estimate similar to 240 telomerase monomers per cell for HEK 293T and HeLa, a number similar to that of telomeres in late S phase. The subunits were in excess of RNPs (e.g. similar to 1150 hTR and similar to 500 hTERT molecules per HeLa cell), suggesting the existence of unassembled components. This hypothesis was tested by overexpressing individual subunits, which increased total telomerase activity as measured by the direct enzyme assay. Thus, there are subpopulations of both hTR and hTERT not assembled into telomerase but capable of being recruited. We also determined the specific activity of endogenous telomerase and of overexpressed super-telomerase both to be similar to 60 nt incorporated per telomerase per minute...

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain
Potisopon, S; Priet, S; Collet, A; Decroly, E; Canard, B; Selisko, B
Nucleic Acids Res. 2014, 42, 11642-11656
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Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel...

Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase
Lohman, GJS; Zhang, YH; Zhelkovsky, AM; Cantor, EJ; Evans, TC
Nucleic Acids Res. 2014, 42, 1831-1844
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Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with k(cat) approximate to 8 x 10(-3) s(-1) and K-M < 1 nM at 25 degrees C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower k(cat) and a K-M approximate to 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl > 100mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 mu M) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase...

A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism
Dy, RL; Przybilski, R; Semeijn, K; Salmond, GPC; Fineran, PC
Nucleic Acids Res. 2014, 42, 4590-4605
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Bacterial abortive infection (Abi) systems are `altruistic' cell death systems that are activated by phage infection and limit viral replication, thereby providing protection to the bacterial population. Here, we have used a novel approach of screening Abi systems as a tool to identify and characterize toxin-antitoxin (TA)-acting Abi systems. We show that AbiE systems are encoded by bicistronic operons and function via a non-interacting (Type IV) bacteriostatic TA mechanism. The abiE operon was negatively autoregulated by the antitoxin, AbiEi, a member of a widespread family of putative transcriptional regulators. AbiEi has an N-terminal winged-helix-turn-helix domain that is required for repression of abiE transcription, and an uncharacterized bi-functional C-terminal domain, which is necessary for transcriptional repression and sufficient for toxin neutralization. The cognate toxin, AbiEii, is a predicted nucleotidyltransferase (NTase) and member of the DNA polymerase beta family. AbiEii specifically bound GTP, and mutations in conserved NTase motifs (I-III) and a newly identified motif (IV), abolished GTP binding and subsequent toxicity...

Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Keen, BA; Jozwiakowski, SK; Bailey, LJ; Bianchi, J; Doherty, AJ
Nucleic Acids Res. 2014, 42, 5830-5845
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PrimPol is a primase-polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol(-/-) cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells...

Direct assessment of transcription fidelity by high-resolution RNA sequencing
Imashimizu, M; Oshima, T; Lubkowska, L; Kashlev, M
Nucleic Acids Res. 2013, 41, 9090-9104
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Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through similar to 10(3) nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity.

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