Kinetic mechanism and fidelity of nick sealing by<i>Escherichia coli</i>NAD<sup>+</sup>-dependent DNA ligase (LigA)

Chauleau, Mathieu; Shuman, Stewart

doi:10.1093/nar/gkw049

Cited by 18 publications

(15 citation statements)

References 57 publications

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“…The 3 0 -remnant must be removed (step 3) by a phosphatase, which in E. coli may be either a function of XthA or Nfo [35] (figure 2). The cleaned onenucleotide gap in DNA is now ready for insertion of the correct dCMP (step 4) by the repair DNA polymerase I (PolA) [39] followed by nick-sealing (step 5) by DNA ligase (LigA) [40]. The excised target and newly inserted residues, along with their corresponding reaction arrows, are shown in red and violet, respectively; dR, deoxyribose.…”

Section: Discussionmentioning

confidence: 99%

Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Alexeeva

Guragain

Tesfahun

et al. 2018

Phil. Trans. R. Soc. B

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Cytosine (C) in DNA is often modified to 5-methylcytosine (mC) to execute important cellular functions. Despite the significance of mC for epigenetic regulation in mammals, damage to mC has received little attention. For instance, almost no studies exist on erroneous methylation of mC by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert mC into ,5-dimethylcytosine (mC) in DNA, m C is probably present We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Nei and Fpg proteins at mC residues The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated mC in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in to initiate its repair.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

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Section: Discussionmentioning

confidence: 99%

Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Alexeeva

Guragain

Tesfahun

et al. 2018

Phil. Trans. R. Soc. B

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“…Since it takes only a single unrepaired double-stand break (DSB) to kill a bacterial cell (47), one problem could be DSBs caused by MutM and MutY incisions at closely spaced lesions (14) or by replication forks encountering unrepaired BER intermediates (45). Other potentially lethal DNA problems include interstrand crosslinks mediated by abasic sites (48), LigA/MutY-dependent futile cycles of ligation/incision (49), and unresolvable collisions caused by stalled transcription or replicative complexes (50).…”

Section: Discussionmentioning

confidence: 99%

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Takahashi

Gruber

Yang

et al. 2017

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

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Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROSdependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ 72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.A gents or conditions that inhibit important biological processes can kill cells. However, physiological processes metabolically downstream of the initial inhibition can also contribute to cell death (1). For example, β-lactam antibiotics not only inhibit penicillin-binding proteins leading to lysis; they also induce a futile cycle of cell wall synthesis and degradation that contributes to their killing (2). In another example, lethal attacks on Escherichia coli mediated by the type VI secretion system P1vir phage and the antimicrobial peptide polymyxin B elicit the production of reactive oxygen species (ROS) that contribute to cell death (3). In bacteria, ROS production has been associated with the lethal effects of diverse stresses (4). In most cases, the detailed mechanisms responsible for ROS production are poorly understood; however, a variety of futile metabolic cycles can elicit H 2 O 2 production, illustrating the breadth of possible metabolic perturbations that could potentially induce oxidative stress (5).Despite widespread evidence that endogenous ROS produced as a consequence of metabolic stress can be lethal to bacteria and eukaryotes, the application of this concept to antibiotic lethality has been complicated. Evidence from multiple investiga...

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“…NAD + -dependent DNA ligases (LigA enzymes), which are ubiquitous in bacteria and essential for bacterial viability in all cases tested, acquired their specificity for NAD + via fusion of an NMN-binding Ia domain module to the N terminus of the NTase domain (4,12,13). Escherichia coli DNA ligase (EcoLigA) was the first cellular DNA ligase discovered and characterized (14)(15)(16)(17)(18)(19)(20), and it remains the premier model for structural and functional studies of the NAD + -dependent DNA ligase family (21)(22)(23)(24)(25)(26)(27). Interest in LigA mechanism is propelled by the promise of targeting LigA (via its signature NAD + substrate specificity and unique structural features vis à vis human DNA ligases) for antibacterial drug discovery (24,28).…”

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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 mechanism and fidelity of nick sealing byEscherichia coliNAD⁺-dependent DNA ligase (LigA)

Cited by 18 publications

References 57 publications

Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

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

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Kinetic mechanism and fidelity of nick sealing byEscherichia coliNAD+-dependent DNA ligase (LigA)

Cited by 18 publications

References 57 publications

Excision of the doubly methylated base N 4 ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Excision of the doubly methylated base N 4 ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

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

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Kinetic mechanism and fidelity of nick sealing byEscherichia coliNAD⁺-dependent DNA ligase (LigA)

Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

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