Figure 1

Alignment and purification of two predicted D. radiodurans DNA ligases. A. Alignment of eight colinear sequence elements in bacterial DNA ligases based on previous studies of DNA ligase motifs [7, 34] using CLUSTALW alignment [35]. The numbers of amino acids between the motifs are indicated. The alignment of motif VI is not shown for the ATP-dependent DNA ligases since the homology is very poor. Note that the putative ATP-dependent DNA ligase from D. radiodurans seems to lack also motif V. The conserved adenylated lysine residue is depicted in bold and labelled with an asterisk. Dr, Deinococcus radiodurans, Ec, Escherichia coli, Bs, Bacillus subtilis, Mt, Mycobacterium tuberculosis, Hi, Haemophilus influenzae. B. Predicted domain structures of D. radiodurans NAD⁺-dependent DNA ligase (LigA) and ATP-dependent DNA ligase (DRB0100). The LigA protein scheme is based on homology searches using the NCBI conserved domain database and the SMART conserved domain database. OB, oligonucleotide-binding fold, Zn, zinc finger, HhH, helixhairpin-helix motif 1, BRCT, BRCA1 C-terminal domain. C. LigA and DRB0100 and their corresponding adenylation mutants LigA K128A and DRB0100 K40A were purified over one metal affinity column and two ion exchange columns to near homogeneity as described in Methods. 3 μg of each protein were loaded onto a 10% SDS-PAGE and the gel was stained with Coomassie Blue R250.