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NAR Top Articles - Nucleic Acid Enzymes

Nucleic Acid Enzymes

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January 2015

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
I. Fonfara, A. Le Rhun, K. Chylinski, K. S. Makarova, A. L. Lecrivain, J. Bzdrenga, E. V. Koonin and E. Charpentier
Nucleic Acids Res. (2014) 42 (4): 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...

RNase L restricts the mobility of engineered retrotransposons in cultured human cells
A. Zhang, B. Dong, A. J. Doucet, J. B. Moldovan, J. V. Moran and R. H. Silverman
Nucleic Acids Res. (2014) 42 (6): 3803-3820
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Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NDelta385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci...

Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli
J. E. Clarke, L. Kime, A. D. Romero and K. J. McDowall
Nucleic Acids Res. (2014) 42 (18): 11733-11751
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Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E...

Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase
V. Gaur, R. Vyas, J. D. Fowler, G. Efthimiopoulos, J. Y. Feng and Z. Suo
Nucleic Acids Res. (2014) 42 (15): 9984-9995
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Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180 degrees with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase...

A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism
R. L. Dy, R. Przybilski, K. Semeijn, G. P. Salmond and P. C. Fineran
Nucleic Acids Res. (2014) 42 (7): 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...

Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA
L. G. Gebhard, J. J. Incicco, C. Smal, M. Gallo, A. V. Gamarnik and S. B. Kaufman
Nucleic Acids Res. (2014) 42 (18): 11668-11686
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Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice...

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain
S. Potisopon, S. Priet, A. Collet, E. Decroly, B. Canard and B. Selisko
Nucleic Acids Res. (2014) 42 (18): 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...

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering
S. Boissel, J. Jarjour, A. Astrakhan, A. Adey, A. Gouble, P. Duchateau, J. Shendure, B. L. Stoddard, M. T. Certo, D. Baker and A. M. Scharenberg
Nucleic Acids Res. (2014) 42 (4): 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.

Binary recombinase systems for high-resolution conditional mutagenesis
M. Hermann, P. Stillhard, H. Wildner, D. Seruggia, V. Kapp, H. Sanchez-Iranzo, N. Mercader, L. Montoliu, H. U. Zeilhofer and P. Pelczar
Nucleic Acids Res. (2014) 42 (6): 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...

Massively parallel determination and modeling of endonuclease substrate specificity
S. B. Thyme, Y. Song, T. J. Brunette, M. D. Szeto, L. Kusak, P. Bradley and D. Baker
Nucleic Acids Res. (2014) 42 (22): 13839-13852
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We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.

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