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

Abstract

NAD+ -dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD + -dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo. D

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).…”

“…To allow biochemical assessment of the activity of this class of enzymes, we wished to determine the ability of a recombinant form of FaLig to join nicks in doublestranded nucleic acids. The gene for FaLig was cloned into the bacterial expression vector pET-16b, overexpressed and purified by affinity chromatography to an in-frame N-terminal His-tag, as has been used in studies of a number of different nucleic acid ligases (Ho and Shuman 2002;Ho et al 2004;Lavesa-Curto et al 2004;Wilkinson et al 2003;Wilkinson et al 2005;Yin et al 2003;Yin et al 2004). Including the addition of the His-tag from pET-16b, the total molecular weight of FaLig is 70.3 kDa.…”