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  • DNA ligase IV contains a conserved

    2020-07-31

    DNA ligase IV contains a conserved ligase domain at its N-terminus and a tandem BRCT domain at its C-terminus (Fig. 1a) [7]. Interaction with Xrcc4 requires the region between the BRCT domains and likely part of the BRCT domain [8], [9]. The DNA ligase IV/Xrcc4 complex is large and its crystal structure has not yet been solved. Structural analysis has relied in part on the use of a range of other ATP-dependent DNA ligases as model systems [10], [11], [12], [13], [14], [15]. The first step of ligation involves the formation of a covalent AMP-enzyme intermediate with AMP being attached to the enzyme via a highly conserved lysine residue. The second step involves the formation of a DNA-adenylate complex followed finally by rejoining. All ATP-dependent DNA ligases have a modular structure of two domains, an adenylation domain (AdD or Domain 1) and an oligo-binding domain (OBD or Domain 2) [12]. Six conserved motifs, designated motifs I, III, IIIa, IV, V and VI, have been identified among covalent nucleotide transferases, of which five are found in the AdD (Fig. 1a). Motifs I, III, IIIa, IV and V are essential for ATP binding and the auto-adenylation reaction. Motif I, encompassing the conserved lysine residue, forms the active site loop of the enzyme and constitutes part of the ATP binding pocket. Based on the crystal structures of a number of DNA ligase complexes, it has been proposed that ligases undergo profound conformational changes upon ATP binding and/or DNA binding [10], [12], [13], [16], [17]. This is exemplified by the rotation of the OBD. Motif VI lies within the OBD, distant from the active site on AdD [10]. However, upon ATP-binding this face of OBD moves towards the active site and residues, including those from motif VI, participate in the adenylation reaction [13], [14], [16], [17]. Subsequently, the OBD moves away from the active site and swivels around placing motif VI far from the AdD, orientating the DNA-binding surface of OBD towards the now adenylated AdD [12], [13], [16], [17]. This switching is essential for the catalytic L002 as it most likely prevents the formation of non-productive complexes between non-adenylated ligase and unnicked/unbroken DNA. The larger eukaryotic ligases, such as LigI and LigIV, also possess an additional N-terminal DNA-binding domain (DBD) that is required for efficient ligation (Fig. 1c) and enables these ligases to encircle DNA [17]. An equivalent helix–hairpin–helix domain is also present in the bacterial NAD-dependent ligases [13], [18]. The toroidal structure of LigI maintains the nicked DNA in a distorted conformation and locates the catalytic domain over the site of the nick prior to ligation [17]. Since DNA ligase IV is essential, the mutations identified in LIG4 syndrome are hypomorphic, that is, they confer residual activity. The analysis of the impact of such mutations is important to help evaluate the clinical impact and potentially to help direct patient care. Additionally, such mutations have the potential to provide novel insight into domains or motifs important for function. In one LIG4 syndrome patient an arginine was mutated to histidine within motif I (R278H) close to the active site lysine residue [19], [20]. More recently, another mutational change (G469E) was identified [5]. Although this mutation lies adjacent to a conserved residue (G468), it lies outside of the six core motifs and, when first identified, we questioned whether it would alter the activity of the enzyme [21]. Our initial studies demonstrated, however, that it was a mutational change that impacted upon function [5]. A sequence comparison between DNA ligases strongly suggests that residues 468–476 represent a further conserved motif present in eukaryotic DNA ligases, which we have designated motif Va (Fig. 1b). The recently reported structure of DNA ligase I also provides evidence that this region might be important for function (Pascal, 2004 #10951). Here, we have undertaken a mutational analysis of residues within motif Va to determine whether it was also important for function in DNA ligase IV. Mutational change of either residue G469 or G468 to glutamic acid markedly reduced protein expression, adenylation and ligation activity. G468A and G469A mutations were better tolerated but were still impacting. Our findings provide biochemical evidence supporting the importance of a glycine at position G468, which is apparent from the DNA ligase I/DNA crystal structure and the high conservation of this residue. Additionally, we found that residues 470–476 do not affect either DNA binding or adenylation activity but significantly impair double-stranded ligation activity. We discuss a model that these residues act, along with another conserved structural motif, as a pincer to facilitate the conformational change of the DNA to enhance catalysis.