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.
Highlights
DNA ligases catalyze the formation of phosphodiester bonds between adjacent 5 P and 3 OH ends in the backbone of double-stranded DNA and have a conserved core structure comprising the catalytic adenylation (AD) domain and an oligonucleotide-binding (OB) domain joined by a flexible linker region [1]
The overall structure of the Alteromonas mediterranea Lig E (Ame-Lig)-DNA complex resolved to 2.3 Aclearly reveals that this Lig E-type ligase binds its substrate without complete encirclement of the DNA duplex (Figure 1)
Inspection of the electron density reveals a phosphodiester bond between the 5 P of the nicked strand and the alpha phosphate of the adenosine monophosphate (AMP), indicating that the step two intermediate has been crystalized after transfer of the cofactor from the catalytic lysine K34 to the DNA (Supplementary Figure S1)
Summary
DNA ligases catalyze the formation of phosphodiester bonds between adjacent 5 P and 3 OH ends in the backbone of double-stranded DNA and have a conserved core structure comprising the catalytic adenylation (AD) domain and an oligonucleotide-binding (OB) domain joined by a flexible linker region [1]. In step 2, the adenosine monophosphate (AMP) moiety is transferred to the 5 phosphate of the nicked DNA activating it for attack by the 3 OH in step 3 forming a new phosphodiester bond that seals the break across the DNA backbone. This catalytic process is accompanied by large-scale reorientations of the AD and OB domains to expose the catalytic site for cofactor and substrate binding and clamp around the DNA during steps 2 and 3 of catalysis [1]. Mutation or deletion of the Chlorella virus ligase latch decreases DNA affinity and activity [4,9]
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