Abstract
DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.
Highlights
The sealing of breaks in the phosphodiester backbone of double-stranded DNA is essential for the replication and survival of all organisms
The nature of the AMP donor for DNA ligases divides them into two classes: the highly conserved NAD-dependent enzymes, which are found in all bacteria and carry out the essential joining of Okazaki fragments during DNA replication (Wilkinson et al, 2001), and the structurally diverse ATP-dependent enzymes, which are found in all domains of life (Martin & MacNeill, 2002)
ATP-dependent DNA ligases share a common catalytic core which includes all six of the conserved nucleotidyltransferase motifs and comprises the adenylation domain (AD domain), where the AMP cofactor is covalently bound, and an oligonucleotide-binding domain (OB domain), doi:10.1107/S1399004714021099 3043 research papers which engages the minor groove of the DNA duplex upon nick binding and assists in the step 1 adenylation reaction (Shuman, 2009; Doherty & Suh, 2000)
Summary
The sealing of breaks in the phosphodiester backbone of double-stranded DNA is essential for the replication and survival of all organisms This function is carried out by DNA ligases in a three-step reaction in which the enzyme first selfadenylates, followed by DNA binding and transfer of AMP to the 50-phosphate terminus of the DNA nick, which is activated for nucleophilic attack by the apposing 30-OH group. These two essential domains are appended with a variety of organism-specific domains and motifs which are involved in DNA binding (Pascal et al, 2004; Nair et al, 2007), cellular localization (Lakshmipathy & Campbell, 1999) or the recruitment of other protein interaction partners (Pascal et al, 2006; Liu et al, 2013; Kiyonari et al, 2006), or in some cases have independent enzymatic functions (Zhu et al, 2006; Zhu & Shuman, 2005, 2006)
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