DNA ligases: progress and prospects

Abstract

DNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates. DNA ligases are essential guardians of genomic integrity, and ligase dysfunction underlies human genetic disease syndromes. Crystal structures of DNA ligases bound to nucleotide and nucleic acid substrates have illuminated how ligase reaction chemistry is catalyzed, how ligases recognize damaged DNA ends, and how protein domain movements and active-site remodeling are used to choreograph the end-joining pathway. Although a shared feature of DNA ligases is their envelopment of the nicked duplex as a C-shaped protein clamp, they accomplish this feat by using remarkably different accessory structural modules and domain topologies. As structural, biochemical, and phylogenetic insights coalesce, we can expect advances on several fronts, including (i) pharmacological targeting of ligases for antibacterial and anticancer therapies and (ii) the discovery and design of new strand-sealing enzymes with unique substrate specificities.

FIGURE 1.

Three-step pathway of nick sealing by DNA ligase.

FIGURE 2.

DNA ligases engage the nicked duplex as C-shaped protein clamps. A and B, ribbon diagrams are shown of the DNA-bound structures of ChVLig (Protein Data Bank code 2Q2T) and HuLig1 (code 1X9N). The proteins were superimposed with respect to their NTase (cyan) and OB (beige) domains. The interdomain contacts that close the ligase clamps are indicated by red arrows. In ChVLig, the latch and NTase domains make kissing contacts. By contrast, HuLig1 closes its clamp via contacts between the N-terminal DBD and the C-terminal OB domain. The peptide linker between the DBD and NTase domain of HuLig1 is indicated by the black arrow. C, the structures of the free ChVLig-AMP intermediate (left; Protein Data Bank code 1FVI) and ligase-AMP bound at a nick (right; with DNA omitted) were superimposed with respect to their NTase domains and then offset laterally to reveal the large movement of the OB domain triggered by nick recognition. The movement entails a rigid body rotation about the hinge segment connecting the NTase and OB domains (indicated by the arrow in free ligase). The surface peptide loop destined to become the latch in the DNA-bound ligase is disordered in the free ligase (dotted line). A sulfate ion is bound on the surface of the free ligase-AMP in a position that mimics the 5′-PO₄ of the nick.

FIGURE 3.

Structural images of LigA bound to nicked DNA, NAD⁺, or a small molecule inhibitor. A, space-filling model of the E. coli LigA protein clamp (Protein Data Bank code 2OWO) encircling nicked duplex DNA, which is rendered as a schematic trace. The modular domain structure of the LigA polypeptide is depicted below the structure. The LigA clamp is closed by kissing contacts between the NTase (cyan) and HhH (beige) domains. B, shown are surface views of the E. faecalis LigA·NAD⁺ complex (upper panel; Protein Data Bank code 1TAE) and the LigA·inhibitor complex (lower panel; code 3BA9) looking into the hydrophobic tunnel of the NTase domain that leads from the surface to the back end of the adenosine-binding pocket. The C-2 atom of adenine is pointing into the tunnel, which is empty in the NAD⁺ complex but filled in the inhibitor complex. C, shown are superimposed structures of NAD⁺ and the pyridopyrimidine inhibitor in their respective LigA complexes. The pyrimidine ring of the inhibitor overlies the adenine base. The bulky pyrido substituent of the inhibitor that fills the tunnel is an offshoot of the equivalent of the adenine C-2 (denoted by the black arrows) and N-3 edge of the ring.