Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis

Abstract

DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct states—an open lysyl-AMP intermediate, and a closed DNA–adenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome.

T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction

“…The deoxyribose moiety of the 5′-terminal nucleotide adjacent to the nick is loosely contacted by Leu458 of T4 DNA ligase, which corresponds to Val383 of E. coli LigA ( 26 ), Phe286 of Chlorella virus ligase ( 25 ), and Phe872 of hLig1 ( 19 ) ( Supplementary Figure S11 ). The aromatic side chain at this position is conserved across all eukaryotic and archaeal DNA ligases, and Phe872 of hLig1, Phe717 of hLig3 ( 22 ) and Phe578 of hLig4 ( 72 ) pack more tightly against the deoxyribose ring than Leu458 of T4 DNA ligase. By contrast, the smaller valine side chain in E. coli LigA leaves a larger gap between the van der Waals radii at this interface.…”

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

“…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).…”

Druggable binding sites in the multicomponent assemblies that characterise DNA double-strand-break repair through non-homologous end joining

“…( B ) DNA Ligase IV zoomed in on conserved residues in the apo state (Ochi et al, 2013; PDB: 3W5O [ 83 ]). ( C ) Molecular surface of the DNA Ligase IV apo state (Ochi et al, 2013; PDB: 3W5O [ 83 ]) ( D ) DNA Ligase IV DNA-bound state (Kaminski et al, 2018; PDB: 6BKG [ 84 ]). ( E ) DNA Ligase IV zoomed in on conserved residues in the DNA-bound state.…”

“…( E ) DNA Ligase IV zoomed in on conserved residues in the DNA-bound state. (Kaminski et al, 2018; PDB: 6BKG [ 84 ]). ( F ) Molecular surface of DNA Ligase IV in the DNA-bound state.…”