Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions

Alexander, Rashada C.; Johnson, Ashley K.; Thorpe, Jeffrey; Gevedon, Travis; Testa, Stephen M.

doi:10.1093/nar/gkg415

Cited by 33 publications

(34 citation statements)

References 42 publications

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“…6D ) are more readily tolerated [31], [34]. In a previous study, nearly all possible combinations of downstream single-nucleotide mismatches yielded >80% ligated product under conditions of low ATP concentrations even with multiple consecutive mismatches [22], [31]. This tolerance for mismatches on the downstream side is sharply contrasted by the results obtained in the present study, where a single tethered nucleotide provides the interaction energy on the downstream side.…”

Section: Discussioncontrasting

confidence: 96%

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

et al. 2010

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Background In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5′ or 2′ OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated.Methodology/Principal FindingsThis study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5′ phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2>A1>A3>A4>A0>A6>A8>A10 and T2>T3>T4>T6≈T1>T8>T10. Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 µM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or T∶G wobble-paired, substrate on the 3′ side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The “universal” analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide.Conclusions/SignificanceOur results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.

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Section: Discussioncontrasting

confidence: 96%

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

et al. 2010

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“…For dbr1Δ whole-cell RNA treated with RNase R and Prp16-IP RNA treated with RNase R, LIT-seq libraries were analyzed similarly, but with several modifications. First, after removal of the adapters, we also removed the two most 3 ′ nucleotides in the 5 ′ library and the two most 5 ′ nucleotides in the 3 ′ library, because the downstream analysis revealed that these nucleotides, which corresponded to the MmeI-derived overhang sequences were frequently mutated, likely due to the tolerance of mismatches in the ligation of a duplex by T4 DNA ligase (Alexander et al 2003); for reasons that are not clear, such mutagenesis was not a significant problem for the RNase R-untreated libraries. After removal of these two nucleotides, reads with 14-15 nt for the 5 ′ libraries and 18-19 nt for the 3 ′ libraries were retained as tags.…”

Section: Bioinformaticsmentioning

confidence: 99%

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Qin

Huang

Wlodaver

et al. 2015

RNA

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Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimalespecially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5 ′ splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3 ′ termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3 ′ to 5 ′ RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

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“…[11] Although the in vivo repair system requires high fidelity for DNA ligation, surprisingly, T4 DNA ligase has been reported to ligate a wide range of substrates such as those containing a gap or one or more mismatches, and even two single-stranded DNAs in the total absence of a template. [12] Moreover, it can variously ligate two DNAs, two RNAs, or even DNA and RNA heteronucleic acids either on a DNA or on an RNA template.…”

Section: Resultsmentioning

confidence: 99%

Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D‐Threoninol

Liang

Fujioka

Asanuma

2011

Chemistry A European J

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Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.

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Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions

Cited by 33 publications

References 42 publications

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D‐Threoninol

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