Abstract
DNA ligases, the enzymes responsible for joining breaks in the phosphodiester backbone of DNA during replication and repair, vary considerably in size and structure. The smallest members of this enzyme class carry out their functions with pared-down protein scaffolds comprising only the core catalytic domains. Here we use sequence similarity network analysis of minimal DNA ligases from all biological super kingdoms, to investigate their evolutionary origins, with a particular focus on bacterial variants. This revealed that bacterial Lig C sequences cluster more closely with Eukaryote and Archaeal ligases, while bacterial Lig E sequences cluster most closely with viral sequences. Further refinement of the latter group delineates a cohesive cluster of canonical Lig E sequences that possess a leader peptide, an exclusively bacteriophage group of T7 DNA ligase homologs and a group with high similarity to the Chlorella virus DNA ligase which includes both bacterial and viral enzymes. The structure and function of the bacterially-encoded Chlorella virus homologs were further investigated by recombinantly producing and characterizing, the ATP-dependent DNA ligase from Burkholderia pseudomallei as well as determining its crystal structure in complex with DNA. This revealed that the enzyme has similar activity characteristics to other ATP-dependent DNA ligases, and significant structural similarity to the eukaryotic virus Chlorella virus including the positioning and DNA contacts of the binding latch region. Analysis of the genomic context of the B. pseudomallei ATP-dependent DNA ligase indicates it is part of a lysogenic bacteriophage present in the B. pseudomallei chromosome representing one likely entry point for the horizontal acquisition of ATP-dependent DNA ligases by bacteria.
Highlights
DNA ligases are essential enzymes in DNA replication and repair, catalyzing the formation of phosphodiester bonds between adjacent 5′P and 3′OH ends in the backbone of double-stranded DNA
Analysis of minimal ATP-dependent DNA ligases by sequence similarity networks revealed that the vast majority of these enzymes are bacterial, despite being less known than the well-characterized viral representatives from Chlorella virus and T7 bacteriophage[12,15]
This is despite the non-essential role and non-ubiquitous distribution of ATP-dependent DNA ligases in bacteria[3,6,19], and can in part be attributed to the predominance of bacterial sequences in the databases relative to eukaryotes, viruses and a rchaea[2]
Summary
DNA ligases are essential enzymes in DNA replication and repair, catalyzing the formation of phosphodiester bonds between adjacent 5′P and 3′OH ends in the backbone of double-stranded DNA They are categorized as being ATP- or NAD-dependent based on the nature of the adenylate cofactor used during catalysis[1,2]. A small sub-set of DNA ligases lack additional globular domains, instead using extended loops or positively-charged binding motifs to engage their DNA substrates[11,12], or relying on recruitment by additional binding partners[13,14] These minimal DNA ligases, all within the ATP-dependent sub-class, include viral ligases from Chlorella virus and T7 bacteriophage which were described in foundational structure–function studies of DNA ligases[12,15]. The purpose is to determine (1) what sequence and potential structural diversity is present in these variants and (2) potential evolutionary trajectories for the differential distribution of these genes among organisms
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