Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Samai, Poulami; Shuman, Stewart

doi:10.1074/jbc.m112.380428

Cited by 14 publications

(16 citation statements)

References 30 publications

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“…In standard ligation buffer (50 mM Tris pH 7.5, 10 mM DTT, 10 mM MgCl 2 , 1 mM ATP), consumption of substrate obeyed apparent saturation kinetics with a constant V 0 /[E] 0 = −0.40 ± 0.03 min − 1 , but resulted in the formation of both AppDNA and ligated product with V 0 /[E] 0 of 0.08 ± 0.02 min − 1 and 0.31 ± 0.02 min − 1 , respectively (for comparison, under identical conditons T4 DNA ligase produced only AppDNA with a rate of 0.0044 ± 0.0006 min − 1 ). When the Mg 2+ concentration was varied (Figure 2A and Supplementary Table S2), it was found that >5 mM MgCl 2 was needed for maximal activity, as expected (43,52). Mn 2+ has previously been shown to substitute for Mg 2+ in the ligation of fully DNA substrates by PBCV-1 DNA ligase with only slight changes to the rate (43).…”

Section: Resultssupporting

confidence: 81%

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Lohman

Zhang

Zhelkovsky

et al. 2013

Nucleic Acids Research

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Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

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Section: Resultssupporting

confidence: 81%

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Lohman

Zhang

Zhelkovsky

et al. 2013

Nucleic Acids Research

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“…Accordingly, it is possible to infer from this information that KREPA1 and KREPA2 stimulate the adenylylation activity of Tb REL2 and Tb REL1 via the undiscovered motif VI in their OB-fold region. Even though, motif VI (RxDK) cannot be readily detected in the sequences of KREPA1 or KREPA2, it has been shown that this motif can exist with insertions [ 31 ]. An alignment between KREPA1 and KREPA2 reveals a conserved region between the two proteins—RxNxK (data not shown) within their predicted OB-fold [ 27 ].…”

Section: Discussionmentioning

confidence: 99%

Mutational Analysis of Trypanosoma brucei RNA Editing Ligase Reveals Regions Critical for Interaction with KREPA2

et al. 2015

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The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1) and the closely related T. brucei RNA Editing Ligase 2 (TbREL2). While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB)-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1) and KREPA1 (for TbREL2). KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441–444) sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.

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“…The fifth metal-bound water bridges to the β phosphate in all three structures. Although we do not have structures of T4 Rnl1 or EcoLigA as the covalent ligase-AMP products of the step 1 adenylylation reaction, the equivalent structure of the covalent NgrRnl-AMP 5 complex at the AMP phosphate (39). Thus, a pentahydrated catalytic metal is likely to be an ancestral feature shared by all modern polynucleotide ligases that use lysyl-AMP intermediates.…”

Section: Discussionmentioning

confidence: 99%

“…Although there are no structures available for a step 1 Michaelis complex of an ATP-dependent DNA ligase, functional studies of Chlorella virus DNA ligase suggest that conserved elements of the C-terminal OB domain are likely candidates to orient the PP i leaving group and coordinate a second metal (38,39).…”

Section: Discussionmentioning

confidence: 99%

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD ⁺ -dependent polynucleotide ligases

Unciuleac

Goldgur

Shuman

2017

Proc. Natl. Acad. Sci. U.S.A.

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Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3′-OH and 5′-PO 4 ). In step 3, ligase catalyzes attack by a DNA or RNA 3′-OH on the polynucleotide-adenylate to seal the two ends via a phosphodiester bond and release AMP. All steps in the ligase pathway require a divalent cation cofactor.The autoadenylylation reaction of polynucleotide ligases is performed by a nucleotidyltransferase (NTase) domain that is conserved in ATP-dependent DNA and RNA ligases and NAD + -dependent DNA ligases (1, 2). The NTase domain includes defining peptide motifs that form the nucleotide-binding pocket. Motif I (KxDG) contains the lysine that becomes covalently attached to the AMP. As Robert Lehman pointed out in 1974 (3), it is unclear how lysine (with a predicted pK a value of ∼10.5) loses its proton at physiological pH to attain the unprotonated state required for attack on the α phosphorus of ATP or NAD + . In principle, a ligase might use a general base to deprotonate the lysine. Alternatively, the pK a could be driven down by positive charge potential of protein amino acids surrounding lysine-Nζ. Several crystal structures of ligases absent metals provided scant support for either explanation. In these structures, the motif I lysine nucleophile is located next to a motif IV glutamate or aspartate side chain (2, 4-6). The lysine and the motif IV carboxylate form an ion pair, the anticipated effect of which is to increase the pK a of lysine by virtue of surrounding negative charge. It is unlikely that a glutamate or aspartate anion could serve as a general base to abstract a proton from the lysine cation. A potential solution to the problem would be if a divalent cation abuts the lysine-Nζ and drives down its pK a .A metal-driven mechanism was revealed by the recent crystal structure of Naegleria gruberi RNA ligase (NgrRnl) as a step 1 Michaelis complex with ATP and manganese (its preferred metal cofactor) (7). The key to capturing the Michaelis-like complex was the replacement of the lysine nucleophile by an isosteric methionine. The 1.9-Å structure contained ATP and two manganese ions in the active site. The "catalytic" metal was coordinated with octahedral geometry to five waters that were, in turn, coordinated by the carboxylate side chains of conserved residues in motifs I, III, and IV. The sixth ligand site in the catalytic metal complex was occupied by an ATP α phosphate oxygen, indicative of a role for the metal in stabilizing the transition state of the autoadenylylation reaction. A key insight, fortified by superposition of the Michaelis complex on the structure of the covalent NgrRnl-(Lys-Nζ)-AMP intermediate, concerned the role of the catalytic metal complex in stabilizing the unprotonated state of the lysine nucleophile before catalysis, via local positive charge and atomic contact of Lys-Nζ to one of the metal-bound waters (7).The NgrRnl Michaelis complex revealed a second metal, coordinated octahedrally to four waters and to ATP β and γ phosphate oxygens. The metal ...

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Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Cited by 14 publications

References 30 publications

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Mutational Analysis of Trypanosoma brucei RNA Editing Ligase Reveals Regions Critical for Interaction with KREPA2

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD ⁺ -dependent polynucleotide ligases

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Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Cited by 14 publications

References 30 publications

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Mutational Analysis of Trypanosoma brucei RNA Editing Ligase Reveals Regions Critical for Interaction with KREPA2

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD + -dependent polynucleotide ligases

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Product

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Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD ⁺ -dependent polynucleotide ligases