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NAR Top Articles - Nucleic Acid Enzymes

Nucleic Acid Enzymes

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December 2013

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity
Mussolino, C; Morbitzer, R; Lutge, F; Dannemann, N; Lahaye, T; Cathomen, T
Nucleic Acids Res. (2011) 39 (21): 9283-9293
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Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that...

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences
Crespan, E; Czabany, T; Maga, G; Hubscher, U
Nucleic Acids Res. (2012) 40 (12): 5577-5590
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'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols beta, lambda and mu, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol lambda and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol beta, not lambda, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol lambda on non-repetitive sequences...

DNA stabilization at the Bacillus subtilis PolX core--a binding model to coordinate polymerase, AP-endonuclease and 3''-5'' exonuclease activities
Banos, B; Villar, L; Salas, M; de Vega, M
Nucleic Acids Res. (2012) 40 (19): 9750-9762
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Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3'-5' exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction...

Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs
Cade, L; Reyon, D; Hwang, WY; Tsai, SQ; Patel, S; Khayter, C; Joung, JK; Sander, JD; Peterson, RT; Yeh, JRJ
Nucleic Acids Res. (2012) 40 (16): 8001-8010
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Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission...

Mechanism of strand displacement synthesis by DNA replicative polymerases
Manosas, M; Spiering, MM; Ding, FY; Bensimon, D; Allemand, JF; Benkovic, SJ; Croquette, V
Nucleic Acids Res. (2012) 40 (13): 6174-6186
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Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases...

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase
Kirpota, OO; Endutkin, AV; Ponomarenko, MP; Ponomarenko, PM; Zharkov, DO; Nevinsky, GA
Nucleic Acids Res. (2011) 39 (11): 4836-4850
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We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10-13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (delta G degrees approximately -6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (delta G degrees approximately -3.3 kcal/mol) together with other specific interactions (delta G degrees approximately -0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the k(cat) term instead; the rate increases by 6-7 orders of magnitude for cognate DNA as compared with non-cognate one. The k(cat) values for substrates of different sequences correlate with the DNA twist...

Antisense RNA associated with biological regulation of a restriction-modification system
Mruk, I; Liu, YP; Ge, LY; Kobayashi, I
Nucleic Acids Res. (2011) 39 (13): 5622-5632
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Restriction-modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (P(REV0)) at the 3' end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the P(M1,M2) promoter. P(M1,M2) promoter-initiated transcription, in turn, appeared to be inhibited by P(REV0). Mutational inactivation of P(REV0) increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in P(REV0) increased restriction of incoming DNA...

Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes
Blower, TR; Short, FL; Rao, F; Mizuguchi, K; Pei, XY; Fineran, PC; Luisi, BF; Salmond, GPC
Nucleic Acids Res. (2012) 40 (13): 6158-6173
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Toxin-antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin-antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin-antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria...

RecA homology search is promoted by mechanical stress along the scanned duplex DNA
Danilowicz, C; Feinstein, E; Conover, A; Coljee, VW; Vlassakis, J; Chan, YL; Bishop, DK; Prentiss, M
Nucleic Acids Res. (2012) 40 (4): 1717-1727
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A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in the dsDNA is extended by strong DNA-protein contacts, whereas the 'complementary' strand is extended by the tension on the base pairs that connect the 'complementary' strand to the 'outgoing' strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.

Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family
Taylor, GK; Heiter, DF; Pietrokovski, S; Stoddard, BL
Nucleic Acids Res. (2011) 39 (22): 9705-9719
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Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 0305 phi 8-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of similar to 60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases...

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