DNA binding with a minimal scaffold: structure–function analysis of Lig E DNA ligases

Abstract

DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5′P and 3′OH ends in an adenylation-dependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker.

Structural insight into DNA joining: from conserved mechanisms to diverse scaffolds

“…The conformation immediately following handover of the covalent linkage of the AMP α-phosphate from the catalytic motif I lysine to the 5′P of the nick ligase-bound DNA-adenylate is captured in the structure of the AD-ligase of Alteromonas mediterranea (Ame-Lig) ( 25 ). Immediately post Step 2, the AMP-DNA phosphoanhydride bond is orientated away from the 3′OH of the nick with the 5′ phosphate of the DNA retaining its original position and the α-phosphate of the AMP forming electrostatic interactions with lysines of motif I and motif V. To be sufficiently close to the 3′OH terminus for nucleophilic attack to occur, the activated phosphate reorients almost 90° about the diphosphate-bond axis, which together with stereochemical inversion during Step 2, returns the AMP nucleoside to a strained syn configuration observed in ligase-DNA adenylate structures from several organisms ( 6 , 25–30 ). This reorientation is mediated by the lysine from motif V as well as the catalytic lysine in motif I, while changes in the non-covalent bonding to the AMP ribose hydroxyls again require rearrangement of contacts to the motif I, III and V residues.…”

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

“…Within a given taxon, two or more DNA ligase enzymes may coexist, often with a division of labor between them. Examples of minimal ATP-dependent ligases include bacteriophage T7 DNA ligase (3), Chlorella virus DNA ligase (4,5), bacterial nonhomologous end-joining (NHEJ) ligases LigD and LigC (6)(7)(8), and bacterial DNA ligase LigE (9,10). The more complex multidomain architectures of ATP-dependent DNA ligases are exemplified by mammalian enzymes Lig1, LigIII, and LigIV (11)(12)(13)(14).…”

Temperature adaptation of DNA ligases from psychrophilic organisms

“…strain SP041 (Psy-Lig) is the smallest DNA ligase that has been structurally studied, being 41 residues shorter than the minimal ChlV-Lig protein . Recent structure-function analysis of Psy-Lig and the closely related Ame-Lig demonstrated a novel mode of ligase engagement with its DNA substrate that relies on well-ordered side-chain contacts on the surface of the conserved domains, rather than re-ordering of flexible loop regions to achieve encirclement of the DNA duplex as was previously observed for minimal viral ligases (Nair et al 2007;Williamson et al 2018). All Lig E-type ADLs have strong predictions for N-terminal leader sequences proposed to direct them to the periplasm.…”

“…The sequence alignment shows that the flexible linker regions connecting the two core domains are similar, preserving the hydrogen bonding pattern observed in Psy-Lig, with the exception of Par-Lig where the equivalent of Lys 176 (Psy-Lig) is replaced by Pro (Fig 3, supplementary figure S1b) . Lig Etype ligases efficiently ligate DNA breaks without any additional DNA-binding domains or large flexible loop regions, instead using interactions with shorter highly structured motifs and specific charged residues found on the DNA-binding surface of the core catalytic domains (Williamson et al 2018;. In general these motifs are well conserved between the three variants, consistent with both the equivalent positively-charged DNA binding surfaces of Vib-Lig and Psy-Lig and previous observations of consensus between Lig Es in this region (Fig 5) (Williamson et al 2018).…”

“…Since the first X-ray crystal structure of an ADL was solved two decades ago from bacteriophage T7 (Subramanya et al 1996), numerous structural analyses of bacterial, archaeal and eukaryotic ADLs have followed (Nishida et al 2006;Pascal et al 2004) (Kim et al 2009;Nishida et al 2006;Pascal et al 2004;Petrova et al 2012) (Akey et al 2006;Kaminski et al 2018;Pascal et al 2006;Shi et al 2018;Williamson et al 2018;, and the wide variety of domains and gene arrangements between the different classes of ligases has become evident. Crystallographic studies of bacteriophage T7 (Doherty and Wigley 1999;Subramanya et al 1996) revealed a common core architecture of two essential catalytic core domains: the adenylation domain (AD) directly involved in catalysis and the site of step 1 enzyme-adenylation, and the smaller oligonucleotide/oligosaccharide binding domain (OB) that is also required for activity (Doherty and Suh 2000;Doherty and Wigley 1999).…”