NAD⁺‐dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor

Abstract

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD ؉ -dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD ؉ as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperaturesensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD ؉ -dependent DNA ligases. Proteins 2003;51:321-326.

Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “Ferroplasma acidarmanus” Fer1

“…Details of bacterial strains and host plasmids used for cloning and protein expression have been described previously (Lavesa-Curto et al 2004;Wilkinson et al 2003;Wilkinson et al 2005). Growth of E. coli was performed at a variety of temperatures on plates and in liquid cultures.…”

“…Cloning of the predicted DNA ligase from F. acidarmanus Fer1 was performed following the strategies employed for bacterial DNA ligases described previously (Lavesa-Curto et al 2004;Wilkinson et al 2003;Wilkinson et al 2005). The gene was amplified by PCR with a proof-reading DNA polymerase from genomic DNA using the following primers: ''Forward primer'': 5¢-CAT ATG ACA AAA TCT TAT AAT ATA CTA TAT G-3¢.…”

“…To allow over-expression of proteins in E. coli GR501, the fulllength gene plus the His-tag were excised from pET16b vectors using the NcoI and BamHI sites and cloned into pTRC99A (Amersham Pharmacia), as described previously (Lavesa-Curto et al 2004;Wilkinson et al 2003;Wilkinson et al 2005).…”

“…In vitro assays of ligation activity were performed using the nicked, 40 bp DNA described previously (LavesaCurto et al 2004;Wilkinson et al 2003;Wilkinson et al 2005). Additional experiments assessed the activity of FaLig with a variety of nicked, 20 bp RNA:DNA hybrids (Bullard and Bowater 2006).…”

“…ATP-dependent DNA ligases and the classical bacterial NAD + -dependent enzymes are distant in evolutionary terms (Nakatani et al 2000). Thus, the amino acid sequences of the NAD + -dependent DNA ligases from E. coli and Streptomyces coelicolor (Wilkinson et al 2003) were used as an out-group to improve confidence in the phylogenetic description. To allow comparison with ATP-dependent DNA ligases from other organisms, sequences from human DNA ligase I and bacteriophages T4 and RB43 were also included.…”