Crystal structure of NAD+-dependent DNA ligase: modular architecture and functional implications

Lee, Jae Young; Chang, Changsoo; Song, Hyun Kyu; Moon, Jinho; Yang, Jong Oh; Kim, Hyun Kyu; Kwon, Seok Joon; Suh, Se Won

doi:10.1093/emboj/19.5.1119

Cited by 170 publications

(197 citation statements)

References 76 publications

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“…LigA resembles a typical bacterial NAD ϩ -dependent ligase; it consists of a core ligase domain flanked by a N-terminal domain (Ia), which is implicated in NAD ϩ recognition (22), and several C-terminal modules, including a tetracysteine zinc-binding motif, a helixhairpin-helix domain, and BRCT domain ( Fig. 1) (23). To evaluate the biochemical properties of MtuLigA, the protein was produced in E. coli as a His 10 -LigA fusion and purified from a soluble extract by adsorption to nickel-agarose resin and elution with buffer containing imidazole ( Fig.…”

Section: Characterization Of M Tuberculosis Liga-m Tuberculosismentioning

confidence: 99%

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

Gong

Martins

Bongiorno

et al. 2004

Journal of Biological Chemistry

129

142

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Mycobacterium tuberculosis encodes an NAD ؉ -dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD ؉ and ATP, respectively, whereas LigC and LigD, which have ATPspecific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA- DNA ligases are grouped into two families, ATP-dependent ligases and NAD ϩ -dependent ligases, according to their nucleotide substrate requirement (1, 2). The ligase reaction entails three nucleotidyl transfer steps (1). In the first step, attack by ligase on the ␣ phosphorus of ATP or NAD ϩ results in release of PP i or NMN and formation of a covalent ligase-adenylate intermediate. 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 a DNA 3Ј-OH on DNA-adenylate to join the two polynucleotides and liberate AMP. ATP-dependent DNA ligases are found in all three domains of life (Bacteria, Archaea, and Eukarya), whereas the NAD ϩ -dependent enzymes are present only in bacteria and entomopoxviruses (1-7).All known bacteria encode a highly conserved NAD ϩ -dependent DNA ligase (LigA). A conditional mutant of Escherichia coli LigA results in growth arrest at the restrictive temperature; thus LigA is essential in E. coli (8,9). LigA is also essential in Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (10 -12). Some bacteria, including E. coli, S. typhimurium, Shigella flexneri, Yersinia pestis, and Pseudomonas putida, have a second NAD ϩ -dependent ligase (13), the function of which is not known.The presumption that bacteria encode only NAD ϩ -dependent DNA ligases was overturned by the demonstration in 1997 of an ATP-dependent ligase in the respiratory pathogen Haemophilus influenzae (5). The 268-amino acid (aa) 1 H. influenzae ligase consists of a minimized catalytic domain. ATP-dependent ligase homologues coexist with NAD ϩ -dependent enzymes in several other bacterial species, including major human pathogens such as Neisseria meningitidis, Y. pestis, Vibrio cholerae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis (3,14,15).Remarkably, M. tuberculosis encodes three distinct ATP-dependent ligase homologues (LigB, LigC, and LigD) plus an NAD ϩ -dependent ligase (LigA) (Fig. 1) (16). To begin to understand the rationale for this plethora of ligases in a single bacterium, we have produced and characterized recombinant versions of the mycobacterial DNA ligase enzymes and probed genetically the essentiality or dispensability of the ATP-dependent ligases for growth of M. tuberculosis. EXPERIMENTAL ...

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Section: Characterization Of M Tuberculosis Liga-m Tuberculosismentioning

confidence: 99%

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

Gong

Martins

Bongiorno

et al. 2004

Journal of Biological Chemistry

129

142

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“…The DNA ligase IV/Xrcc4 complex is large and its crystal structure has not yet been solved. Structural analysis has relied in part on the use of a range of other ATPdependent DNA ligases as model systems [10][11][12][13][14][15]. The first step of ligation involves the formation of a covalent AMP-enzyme intermediate with AMP being attached to the enzyme via a highly conserved lysine residue.…”

Section: Introductionmentioning

confidence: 99%

“…Based on the crystal structures of a number of DNA ligase complexes, it has been proposed that ligases undergo profound conformational changes upon ATP binding and/or DNA binding [10,12,13,16,17]. This is exemplified by the rotation of the OBD.…”

Section: Introductionmentioning

confidence: 99%

“…However, upon ATPbinding this face of OBD moves towards the active site and residues, including those from motif VI, participate in the adenylation reaction [13,14,16,17]. Subsequently, the OBD moves away from the active site and swivels around placing motif VI far from the AdD, orientating the DNA-binding surface of OBD towards the now adenylated AdD [12,13,16,17]. This switching is essential for the catalytic cycle as it most likely prevents the formation of non-productive complexes between non-adenylated ligase and unnicked/unbroken DNA.…”

Section: Introductionmentioning

confidence: 99%

“…1c) and enables these ligases to encircle DNA [17]. An equivalent helixhairpin-helix domain is also present in the bacterial NAD-dependent ligases [13,18].…”

Section: Introductionmentioning

confidence: 99%

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Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

et al. 2006

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Penny (2006) Identification of a novel motif in DNA ligases exemplified by DNA ligase IV. DNA Repair, 5 (7). pp. 788-798. ISSN 1568-7864 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/17805/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the URL above for details on accessing the published version. Copyright and reuse:Sussex Research Online is a digital repository of the research output of the University.Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available.Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly however, such mutational changes do impact upon double stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggests that motif Va functions as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.3

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Structural Zinc Sites

Auld

2004

Handbook of Metalloproteins

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Structural zinc binding sites in proteins have a combination of four nitrogen, oxygen, and sulfur ligands provided by the side chains of amino acids of the protein. In marked contrast to catalytic zinc sites, no metal‐coordinated water molecule is present. There are over seven dozen representatives of structural zinc sites. While the sulfur of a cysteine is by far the preferred ligand for such sites, it is also found in combination with an imidazole nitrogen of a histidine, and/or a carboxylate oxygen from aspartate or glutamate. Of the 22 possible permutations of these 4 ligands, 8 have been observed. The ligands to these metal sites are provided by protein sequences as small as 10 amino acids and as large as 210 amino acids but the majority fall within the range of 25 to 50 amino acids. The ligands are frequently supplied from within or just before or after a β‐sheet secondary structure. The role of a structural zinc site is probably twofold. It maintains the structure of the protein in the immediate vicinity of the metal site. In this manner, it may influence enzymatic activity by providing active site residues involved in catalysis and/or affecting the chemical environment of catalytic groups through interaction with amino acids originating from within its metal‐binding spacers. However, in many cases these metal sites also affect the overall stability of the protein as judged by temperature and pH criteria.

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Crystal structure of NAD+-dependent DNA ligase: modular architecture and functional implications

Cited by 170 publications

References 76 publications

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

Structural Zinc Sites

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