Chlorella virus DNA ligase: nick recognition and mutational analysis

Sriskanda, Verl

doi:10.1093/nar/26.2.525

Cited by 69 publications

(76 citation statements)

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“…The K481A protein was also able to seal the nicked DNA-adenylylate, demonstrating that the motif I lysine nucleophile is not essential for phosphodiester bond formation by LigD. This result agrees with similar findings concerning the motif I lysines of vaccinia, Chlorella virus, and Methanobacterium DNA ligases (12,52,57).…”

Section: Mutational Effects On the Ligase Adenylylation Reaction-the supporting

confidence: 89%

“…This side chain is conspicuously not required for step 1 adenylylation in the case of Chlorella virus DNA ligase (4,12), human Lig1 (51), Methanobacterium DNA ligase (52), T4 RNA ligase 1 (53), Thermus thermophilus DNA ligase (54), or E. coli LigA (16) but is instead essential during downstream steps of the ligation reactions catalyzed by those enzymes. In the case of RNA capping enzymes, the motif I aspartate, although conserved, is not required for enzyme guanylylation in vitro or the composite RNA capping reaction in vivo (19,55,56).…”

Section: Mutational Effects On the Ligase Adenylylation Reaction-the mentioning

confidence: 99%

“…Structure-function analyses of exemplary ligases and capping enzymes have pinpointed five conserved peptide motifs (see Fig. 1, I, III, IIIa, IV, and V ) within the NT domain that comprise the NMP-binding pocket (12)(13)(14)(15)(16)(17). The OB domain of ATP-dependent DNA ligases and mRNA capping enzymes contains motif VI, which coordinates the PP i leaving group and is critical for the enzyme nucleotidylylation step (6,(17)(18)(19).…”

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Crystal Structure and Nonhomologous End-joining Function of the Ligase Component of Mycobacterium DNA Ligase D

Akey

Martins

Aniukwu

et al. 2006

Journal of Biological Chemistry

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DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 Å crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3-OH and caged by arginine and lysine side chains is a putative mimetic of the 5-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in ⌬ligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.DNA ligases are essential for the maintenance of genome integrity. As nick-sealing enzymes, they are responsible for joining the Okazaki fragments on the lagging strand of the DNA replication fork and for restoring the continuity of the DNA backbone subsequent to nucleotide excision and base excision repair. Nick sealing entails a series of three nucleotidyl transfer reactions (1). In the first step, a lysine nucleophile in the ligase active site attacks the ␣-phosphorus of ATP or NAD ϩ to form a covalent lysyl-N-AMP intermediate and expel either pyrophosphate or nicotinamide mononucleotide. In the second step, the 5Ј-phosphate of the DNA nick attacks the phosphorus of lysyl-AMP to form a DNAadenylylate (A(5Ј)pp(5Ј)DNA) intermediate and expel the lysine. The third step is the attack of the nick 3Ј-OH on the DNA-adenylylate to form a 3Ј-5Ј-phosphodiester and release AMP. DNA ligases comprise one branch of the covalent nucleotidyltransferase superfamily, which includes RNA ligases and mRNA capping enzymes (2). The superfamily members catalyze chemically similar NMP addition reactions at polynucleotide 5Ј ends via lysyl-N-NMP intermediates. DNA ligases and mRNA capping enzymes contain two structural modules that compose a catalytic core, a proximal nucleotidyltransferase (NT) 4 domain and a distal OB fold (OB) domain (3-11). Structure-function analyses of exemplary ligases and capping enzymes have pinpointed five conserved peptide motifs (see Fig. 1, I, III, IIIa, IV, and V ) within the NT domain that comprise the NMP-binding pocket (12)(13)(14)(15)(16)(17). The OB domain of ATP-dependent DNA ligases and mRNA capping enzymes contains motif VI, which coordinates the PP i leaving group and is critical for the enzyme nucleotidylylation step (6,(17)(18)(19).Many organisms...

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Section: Mutational Effects On the Ligase Adenylylation Reaction-the supporting

confidence: 89%

Section: Mutational Effects On the Ligase Adenylylation Reaction-the mentioning

confidence: 99%

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confidence: 99%

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Crystal Structure and Nonhomologous End-joining Function of the Ligase Component of Mycobacterium DNA Ligase D

Akey

Martins

Aniukwu

et al. 2006

Journal of Biological Chemistry

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“…The sealing reactions were initiated by adding ligase. Aliquots (20 l) were withdrawn at the times specified and quenched immediately with EDTA and formamide (12).…”

Section: Methodsmentioning

confidence: 99%

Conserved Residues in Domain Ia Are Required for the Reaction of Escherichia coli DNA Ligase with NAD+

Sriskanda

Shuman

2002

Journal of Biological Chemistry

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NAD؉ -dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD ؉ ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD ؉ and then to isolate small molecules that recognize these components and thereby block the binding of NAD ؉ to the ligase. The limitation to this strategy is that the structural determinants of NAD ؉ specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD ؉ requires N-terminal domain Ia, which is unique to, and conserved among, NAD ؉ ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3-OH/5-PO 4 nicks and the reaction with NAD؉ to form ligaseadenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5-PO 4 nick and adenylyl transfer from NAD ؉ without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD ؉ . Structure-activity relationships were clarified by conservative substitutions. DNA ligases are grouped into two families, ATP-dependent ligases and NADϩ -dependent ligases, according to the cofactor required for ligase-adenylate formation (1). Both types of DNA ligases catalyze the sealing of 5Ј-phosphate and 3Ј-hydroxyl termini at nicks in duplex DNA by means of three sequential nucleotidyl transfer reactions. In the first step, attack on the ␣ phosphorus of ATP or NAD ϩ by ligase results in release of pyrophosphate or nicotinamide mononucleotide (NMN) 1 and formation of a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide bond to lysine. In the second step, the AMP is transferred to the 5Ј end of the 5Ј-phosphate-terminated DNA strand to form DNA-adenylate (AppDNA). In the third step, ligase catalyzes attack by the 3Ј-OH of the nick on DNA-adenylate to join the two polynucleotides and release AMP.

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“…It is noteworthy that whereas conservative mutations of the motif I aspartate of T4 Rnl1 (to Asn, Ser, or Glu) also had no effect on step 1, such changes abolished step 2 and step 3 of the ligation pathway (18). The motif I Asp is essential for step 2 catalysis by Chlorella virus DNA ligase and is also implicated in step 3 (30,31). It would seem that Rnl2 is less reliant on this motif I side chain for catalysis of step 2 than is Rnl1 and DNA ligase.…”

Section: Mutational Analysis Of Rnl2 Identifies Residues Essential Fomentioning

confidence: 97%

Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains

Shuman²

2002

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

147

174

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RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain-a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.T 4 RNA ligase (1) is the founding member of a family of RNA end-joining enzymes involved in RNA repair, splicing, and editing pathways (2-8). T4 RNA ligase joins 3Ј OH and 5Ј PO 4 RNA termini by means of three nucleotidyl transfer steps similar to those of DNA ligases (9-11).Step 1 is the reaction of RNA ligase with ATP to form a covalent ligase-(lysyl-N)-AMP intermediate. In step 2, the AMP is transferred to a 5Ј PO 4 RNA end to form an RNA-adenylate intermediate (AppRNA). In step 3, attack by an RNA 3Ј OH on RNA-adenylate seals the ends and releases AMP. T4 RNA ligase can join two single-stranded RNA molecules without a complementary bridging polynucleotide. T4 RNA ligase can also catalyze intramolecular ligation to form a covalently closed RNA circle. RNA ligase has been a powerful tool in the synthesis of RNAs of defined sequence, RNA 3Ј end modification, RNA 3Ј end-labeling, RNA sequencing, and structural analysis (11).During T4 infection in vivo, the RNA ligase, together with T4 polynucleotide kinase (Pnk), performs an RNA repair function that remodels and then seals broken tRNA ends. In Escherichia coli strains containing the prr locus, the host cell tRNA Lys is cleaved 5Ј to the wobble position by a T4-induced anticodon nuclease. If Pnk and RNA ligase are not present, the synthesis of viral proteins is blocked by depletion of tRNA Lys , and the phage cannot replicate (2,(12)(13)(14). This pathway represents an RNA-based restriction system of host defense against a foreign invader. tRNA anticodon nuclease systems are present in other pathogenic bacteria (14). The bacterial toxins colicins D and E5 are also anticodon nucleases that attack specific tRNAs (15). Thus, RNA repair enzymology has broad significance as a means to combat, or reco...

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Chlorella virus DNA ligase: nick recognition and mutational analysis

Abstract: ABSTRACT

Cited by 69 publications

References 1 publication

Crystal Structure and Nonhomologous End-joining Function of the Ligase Component of Mycobacterium DNA Ligase D

Crystal Structure and Nonhomologous End-joining Function of the Ligase Component of Mycobacterium DNA Ligase D

Conserved Residues in Domain Ia Are Required for the Reaction of Escherichia coli DNA Ligase with NAD+

Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains

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