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  • In contrast to G and G which

    2019-07-18

    In pool to G468 and G469, which are buried (Fig. 7a), residues 470–476 are equivalent to the surface loop L12 present in the crystal structure of the LigI complex (Fig. 7b) [17]. The OBD of DNA ligases adopts at least two distinct orientations during the catalytic cycle. In the adenylation step, the face of the OBD which contains motif VI swivels towards the AdD following ATP binding, acting as a “lid” over the active site, with a number of OBD residues directly participating in the adenylation reaction. In the DNA binding/ligation step, the OBD lid opens up (following adenylation) and the domain pivots around, orienting the DNA binding face including motif Va towards the active site, positioning motif VI away from the AdD. The OBD of LigI binds directly to DNA [17] interacting with the minor groove adjacent to the nicked DNA (Fig. 7b). Pascal et al. have reported that the OBD alters the curvature of the DNA backbone, conferring an A-form conformation (with an expanded minor groove) on the DNA upstream of the nick and maintaining a B-form on the downstream side [17]. A number of regions of LigI are critical for preserving the B-type DNA conformation including the loops L12, L45 and α-helix S. Surface loop L12 (motif Va) binds to the template strand of DNA (Fig. 7b), making direct contacts via a number of residues, including Arg771 (R476 in Lig IV) that extends from L12 into the minor groove (Fig. 7b). Our findings reveal that residues in the loop region of motif Va (470–476) do not impact upon adenylation but are required for double-strand ligation activity. Based on the crystal structure of Lig I, Motif Va loop was suggested to function in conjunction with helix S that binds to the opposite strand, as a molecular pincer that senses the dimensions of the minor groove (Fig. 7b) and actively maintains the B-form that is essential for efficient ligation. Consistent with this model, it has been shown that the presence of an A-form structure downstream of a nick severely affects ligation efficiency [17], [23], [24]. Our findings provide biochemical evidence for this model. Although residues 470–476 may be important for modulating the conformation of the DNA, they do not appear to be critical for DNA binding, which is likely due to the fact that the DNA makes many contacts with DNA ligase IV involving all three domains [17]. Interestingly, a mutation A771W in DNA ligase I has been described in a patient [25]. A771 lies within the equivalent motif in ligase I. This mutation does not affect adenylate complex formation but does affect nick ligation consistent with a molecular pincer model [26]. Notably, mice expressing this mutation exhibit an increased predisposition to cancer [27]. Interestingly, although DNA ligases have different substrate specificities and DNA ligase III and IV can rejoin DNA/RNA substrates in contrast to DNA ligase I, residues 470–476 are reasonably well conserved between all DNA ligases, suggesting a role distinct from substrate specificity. Nevertheless, differences in this region between ligases might relate to substrate specificity.
    Acknowledgements
    Introduction DNA ligases play an important role in genome replication and DNA damage repair. DNA ligases are classified into two families according to their nucleotide substrate requirement: ATP-dependent ligases and NAD-dependent ligases [1], [2]. The ligase reaction consists of three nucleotidyl transfer steps [1]. In the first step, ligase attacks the phosphorus of ATP or NAD, resulting in the release of PPi or NMN and formation of a covalent ligase-adenylate intermediate. In the second step, the AMP is transferred to the 5′ end of the 5′ phosphate-terminated DNA strand to form DNA-adenylate. In the third step, ligase catalyzes the formation of a phosphodiester bond between 3′-OH and 5′ phosphate, resulting in the release of AMP. ATP-dependent DNA ligases are found in all three domains of life (bacteria, archaea, and eukarya), whereas NAD-dependent ligases are present only in bacteria and entomopoxviruses [1], [2], [3], [4], [5].