title: Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.
abstract: The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.
authors: Division of Parasitology, Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
authors: Nidhi
authors: N
authors: Shrivastava
authors: Jeetendra Kumar
authors: JK
authors: Nag
authors: Shailja
authors: S
authors: Misra-Bhattacharya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D017178:Brugia malayi / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D011088:DNA Ligases / Q000737:chemistry*; D005260:Female; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods; D020577:Wolbachia / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D011088:DNA Ligases; C120640:DNA ligase (NAD)