Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase

Samai, Poulami; Shuman, Stewart

doi:10.1074/jbc.m111.226191

Cited by 11 publications

(31 citation statements)

References 27 publications

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“…Therefore, it is interesting to compare the ligation activity of human LIG1, which is one of the largest ligases, with the ligase from Chlorella virus, which is one of the smallest ligases. Recent pre-steady state analysis of the Chlorella virus DNA ligase revealed almost identical rate constants for adenylyl transfer and nick sealing as we have observed for human LIG1 (21). Consistent with their similar kinetic param- eters in vitro, both enzymes are able to rescue the growth of yeast lacking cdc9, the replicative DNA ligase (22,23).…”

supporting

confidence: 60%

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Taylor

Conrad

Wahl

et al. 2011

Journal of Biological Chemistry

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DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg 2؉ ions for catalysis and that an essential Mg 2؉ ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg 2؉ changes along the reaction coordinate. At saturating concentrations of ATP and Mg 2؉ ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg 2؉ , the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.Breaks in DNA result from spontaneous hydrolysis of the phosphodiester backbone and are formed as transient intermediates during DNA replication and repair pathways. Mammals have three genes encoding DNA ligases that seal these breaks and restore the continuous nature of chromosomes. DNA ligase I (LIG1) 2 is essential for ligation of single-strand breaks in the nucleus, including the ligation of Okazaki fragments during discontinuous DNA replication (1). DNA ligase III (LIG3) is required for mitochondrial DNA replication and repair. Although LIG3 has been assumed to play essential roles in nuclear DNA repair, it was recently shown to be dispensable for nuclear genomic maintenance (2, 3). DNA ligase IV (LIG4) is specialized for repair of nuclear double-strand breaks and is required for nonhomologous end joining and V(D)J recombination (4, 5).The overall reaction catalyzed by DNA ligases involves the formation of a phosphodiester bond between an adjacent 3Ј-hydroxyl and a 5Ј-phosphate in DNA. The reaction proceeds via a universally conserved pathway ( Fig. 1) (6 -8). First, the apoenzyme catalyzes transfer of the AMP group from a nucleotide cofactor to an active site lysine, forming an adenylylated enzyme intermediate. Eukaryotic ligases use ATP as the adenylyl group donor, whereas bacterial ligases utilize either ATP or NAD ϩ . After binding a nicked DNA substrate, the adenylylated enzyme catalyzes transfer of the adenylyl group to the 5Ј-phosphate present at the nick, forming an adenylylated DNA intermediate. In the fina...

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supporting

confidence: 60%

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Taylor

Conrad

Wahl

et al. 2011

Journal of Biological Chemistry

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“…Experience with classic DNA ligases teaches that the A(5′)pp(5′) DNA intermediate can be difficult to detect under optimal ligation reaction conditions, because the rate of the phosphodiester synthesis step is much faster than the rate of A(5′)pp(5′) DNA formation (20). However, the abundance and persistence of the adenylylated polynucleotide in the classic ligase scheme can be enhanced by nucleic acid modifications that slow the final step (21).…”

mentioning

confidence: 99%

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

Chakravarty

Subbotin²,

Chait³

et al. 2012

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

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105

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A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′ phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5′)pp(5′)N-in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3′-phosphate and 5′-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3′-phosphate to form a polynucleotide-(3′)pp(5′)G intermediate; and (iii) attack of a 5′-OH on the -N(3′)pp(5′)G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3′ ends. RNA or A(5′)pp(5′)DNA; and (iii) ligase catalyzes attack by a polynucleotide 3′-OH on A(5′)pp(5′)RNA/DNA to form a 3′-5′ phosphodiester bond and release AMP (1). The salient principle of classic ligase catalysis is that the high energy of the ATP phosphoanhydride bond is transferred via the enzyme to the polynucleotide 5′ end, thereby activating the 5′ end for phosphodiester synthesis, with AMP as a favorable leaving group. The shared chemical mechanism of classic RNA and DNA ligases is reflected in their conserved core tertiary structures and active sites (1).Classic ATP-dependent RNA ligases are encoded by diverse taxa in all three phylogenetic domains. In bacteria, fungi, and plants they function as components of multienzyme RNA repair pathways that heal and seal broken RNAs with 2′,3′ cyclic phosphate and 5′-OH ends (2-5). In the healing phase, the 2′,3′ cyclic phosphate end is hydrolyzed by a phosphoesterase enzyme to a 3′-OH, and the 5′-OH end is phosphorylated by a polynucleotide kinase enzyme to yield a 5′-monophosphate. The resulting 3′-OH and 5′-phosphate ends are then suitable for ATP-dependent sealing by RNA ligase. This "healing-and-sealing" pathway is responsible for tRNA splicing in fungi and plants (6, 7), for mRNA splicing in the fungal unfolded protein response (8), and for tRNA restriction repair during bacteriophage infection of Escherichia coli (9).An alternative "direct ligation" pathway for joining 2′,3′ cyclic phosphate and 5′-OH ends during mammalian tRNA splicing was discovered nearly 30 y ago (10-12), when it was shown that the 2′,3′ cyclic phosphate of the cleaved pre-tRNA is incorporated at the splice junction in the mature tRNA. (The junction phosphate in yeast tRNA splicing derives from the γ-phosphate of the NTP substrate for the 5′ kinase step.) Direct ligation languished until 2011, when three laboratories identified bacterial, archaeal, and mammalian RtcB proteins as RNA ligase enzymes capable of seali...

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“…1B for the 3 ′ C:I substrate), with each datum being the average of three independent experiments. The kinetic profiles were fitted in MATLAB to a unidirectional scheme of 5 ′ -PO 4 adenylylation (step 2: Rnl2-AMP • pNick → Rnl2 • AppNick) and subsequent phosphodiester synthesis (step 3; Rnl2 • AppNick → Rnl2 • AMP • RNApDNA), with allowance for reversible branching of Rnl2 • AppNick to an "out-of-pathway" state, as described in our prior studies of Chlorella virus DNA ligase (Samai and Shuman 2011a). The observed step 2 and step 3 rate constants (k step2 and k step3 ) for the series of correctly paired 3 ′ N:X nicks are shown in Figure 1C.…”

Section: Resultsmentioning

confidence: 99%

“…The distributions of AppDNA intermediate and sealed product were plotted as a function of reaction time. As noted previously for Chlorella virus DNA ligase (Samai and Shuman 2011a), our initial modeling of the data in Prism to a simple sequential reaction pathway described by two rate constants-for 5 ′ -adenylylation (step 2) and phosphodiester synthesis (step 3)-yielded poor fits to the experimental kinetic profiles for the 5 ′ -adenylylated intermediate, the level of which typically declined more slowly than could be simulated by the simple sequential pathway. To better model the experimental profiles for AppDNA and sealed product, we used the kinetic scheme described for Chlorella virus DNA ligase (Samai and Shuman 2011a), which includes a reversible transition of the step 2 product from an active "in pathway" state to an inactive "out of pathway" state.…”

Section: Kinetics Of Single-turnover Nick Sealingmentioning

confidence: 99%

“…As noted previously for Chlorella virus DNA ligase (Samai and Shuman 2011a), our initial modeling of the data in Prism to a simple sequential reaction pathway described by two rate constants-for 5 ′ -adenylylation (step 2) and phosphodiester synthesis (step 3)-yielded poor fits to the experimental kinetic profiles for the 5 ′ -adenylylated intermediate, the level of which typically declined more slowly than could be simulated by the simple sequential pathway. To better model the experimental profiles for AppDNA and sealed product, we used the kinetic scheme described for Chlorella virus DNA ligase (Samai and Shuman 2011a), which includes a reversible transition of the step 2 product from an active "in pathway" state to an inactive "out of pathway" state. Simulations and curve fitting to the experimental data to this model were performed in MATLAB by inputting an initial k step2 value (obtained by plotting the sum of AppDNA plus sealed RNApDNA as a function of time and fitting the data to a single exponential in Prism) and stipulating that all other rates be greater than zero.…”

Section: Kinetics Of Single-turnover Nick Sealingmentioning

confidence: 99%

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Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

Chauleau

Shuman

2013

RNA

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′ -OH nick termini, k step2 was fast (9.5 to 17.9 sec −1 ) and similar in magnitude to k step3 (7.9 to 32 sec −1 ). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3 ′ -OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3 ′ -OH elicited severe decrements in the rate of 5 ′ -adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3 ′ -terminal ribonucleoside at the nick for optimal 5 ′ -adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.

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Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase

Cited by 11 publications

References 27 publications

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

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