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NAR Top Articles - Nucleic Acid Enzymes

Nucleic Acid Enzymes

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September 2015

The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor
Gossan, NC; Zhang, F; Guo, BQ; Jin, D; Yoshitane, H; Yao, AY; Glossop, N; Zhang, YQ; Fukada, Y; Meng, QJ
Nucleic Acids Res. 2014, 42, 5765-5775
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Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A...

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...

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...

Replisome mechanics: lagging strand events that influence speed and processivity
Georgescu, RE; Yao, NN; Indiani, C; Yurieva, O; O'Donnell, ME
Nucleic Acids Res. 2014, 42, 6497-6510
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The antiparallel structure of DNA requires lagging strand synthesis to proceed in the opposite direction of the replication fork. This imposes unique events that occur only on the lagging strand, such as primase binding to DnaB helicase, RNA synthesis, and SS B antigen (SSB) displacement during Okazaki fragment extension. Single-molecule and ensemble techniques are combined to examine the effect of lagging strand events on the Escherichia coli replisome rate and processivity. We find that primase activity lowers replisome processivity but only when lagging strand extension is inoperative. rNTPs also lower replisome processivity. However, the negative effects of primase and rNTPs on processivity are overcome by the extra grip on DNA provided by the lagging strand polymerases. Visualization of single molecules reveals that SSB accumulates at forks and may wrap extensive amounts of single-strand DNA. Interestingly SSB has an inter-strand positive effect on the rate of the leading strand based in its interaction with the replicase chi-subunit. Further, the lagging strand polymerase is faster than leading strand synthesis, indicating that replisome rate is limited by the helicase...

Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution
El Omari, K; Meier, C; Kainov, D; Sutton, G; Grimes, JM; Poranen, MM; Bamford, DH; Tuma, R; Stuart, DI; Mancini, EJ
Nucleic Acids Res. 2013, 41, 9396-9410
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Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, phi 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from phi 6, phi 8 and phi 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in phi 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops...

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.

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...

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...

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.

In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity
Wang, MQ; Zhang, HF; Zhang, W; Zhao, YY; Yasmeen, A; Zhou, L; Yu, XQ; Tang, Z
Nucleic Acids Res. 2014, 42, 9262-9269
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Single-nucleotide polymorphisms, either inherited or due to spontaneous DNA damage, are associated with numerous diseases. Developing tools for site-specific nucleotide modification may one day provide a way to alter disease polymorphisms. Here, we describe the in vitro selection and characterization of a new deoxyribozyme called F-8, which catalyzes nucleotide excision specifically at thymidine. Cleavage by F-8 generates 3'- and 5'-phosphate ends recognized by DNA modifying enzymes, which repair the targeted deoxyribonucleotide while maintaining the integrity of the rest of the sequence. These results illustrate the potential of DNAzymes as tools for DNA manipulation.

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