NAR Top Articles - Nucleic Acid Enzymes
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
Free Full Text
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...
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
Free Full Text
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...
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
Free Full Text
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...
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
Free Full Text
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...
Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence
Schauer, GD; Huber, KD; Leuba, SH; Sluis-Cremer, N
Nucleic Acids Res. 2014, 42, 11687-11696
Free Full Text
Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are routinely used to treat HIV-1 infection, yet their mechanism of action remains unclear despite intensive investigation. In this study, we developed complementary single-molecule fluorescence and ensemble fluorescence anisotropy approaches to discover how NNRTIs modulate the intra-molecular conformational changes and intermolecular dynamics of RT-template/primer (T/P) and RT-T/P-dNTP complexes. We found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity. Further, efavirenz promotes formation of the E138-K101 salt bridge between the p51 and p66 subunits of RT, which contributes to opening of the thumb/fingers subdomains. Engineering a more polar salt bridge between p51 and p66 resulted in even greater increases in the thumb/fingers opening, RT sliding, dNTP binding disruption and in vitro and in vivo RT inhibition than were observed with wild-type RT...
Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA
Zhang, JC; Zhou, RB; Inoue, J; Mikawa, T; Ha, T
Nucleic Acids Res. 2014, 42, 3821-3832
Free Full Text
Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20-500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins...
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
Free Full Text
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...
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
Free Full Text
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.
Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli
Clarke, JE; Kime, L; Romero, AD; McDowall, KJ
Nucleic Acids Res. 2014, 42, 11733-11751
Free Full Text
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...
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
Free Full Text
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.
- About this journal
- NAR Methods online
- 2015 Database Issue
- 2014 Web Server Issue
- NAR Special Collections
- Referee Information
- Rights & Permissions
- Dispatch date of the next issue
- This journal is a member of the Committee on Publication Ethics (COPE)
- view Recent Comments on articles
- We are mobile – find out more
- Journals Career Network
Impact factor: 8.808
5-Yr impact factor: 8.378
Senior Executive Editors
- Instructions to authors
- Scope and Criteria for Consideration
- Submit a manuscript now
- Self-archiving policy
Open access options for authors