Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis

Abstract

The crystal structure of NAD+-dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1-581), Tfi-M2 (residues 1-448), Tfi-M3 (residues 1-403) and Tfi-M4 (residues 1-314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme-AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme-DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wild-type. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nick-closing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type.

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

“…2C). This is in good agreement with recent observations for similar fragments from other NAD + -ligases [29,37]. Additional preliminary experiments confirmed that LigA lacking the BRCT domain (DBRCT-lig) could join double-strand breaks in plasmids, though at a much reduced rate compared to full LigA (data not shown).…”

“…As discussed above, the detection of DNA binding by the BRCT domain of E. coli LigA confirms recent observations using LigA from thermophilic bacteria [29,37]. These studies are also consistent with previous analyses of NAD + -ligases from B. stearothermophilus and S. aureus, which showed that stable DNA binding was provided by a C-terminal fragment that contains the BRCT domain [8,31].…”

“…Therefore, these regions may require the presence of other regions to stabilise their interaction with DNA. This may be due to domain Ia [15], but the BRCT domain may also be involved in forming stable protein-DNA complexes, as suggested by the current data and that obtained recently using other NAD + -ligases [29,37]. It is clear that additional high-resolution studies are required to resolve the molecular details of DNA binding to NAD + -ligases.…”

“…4A). This is in contrast to recent observations that used much higher protein:DNA amounts (N100:1) for similar fragments from other NAD + -ligases [29,37]. To evaluate the differences between these results, full-length LigA and DBRCT-lig were tested for their ability to bind DNA over a range of different protein concentrations (Fig.…”

NAD+-dependent DNA ligase (Rv3014c) from M. tuberculosis: Strategies for inhibitor design

“…More work is necessary to actually elucidate the role of this domain in detail. Its deletion resulted in threefold reduction in activity of E. coli LigA (Wilkinson et al, 2005), whereas the T. filiformis LigA retains activity even after its deletion (Jeon et al, 2004). In contrast, no activity, either in vitro or in vivo, is observed in the case of the M. tuberculosis enzyme.…”