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.
Highlights
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
LigIV catalyzes the ligation step during repair of double-strand break (DSB) through classical nonhomologous end-joining (NHEJ)[7] and in V(D)J recombination that is critical for immunoglobulin gene maturation[8]
To better understand the unique features of LigIV, we obtained two crystal structures of protein-DNA complexes in the ligation pathway—the lysyl-AMP intermediate in an open conformation, and the DNA–adenylate in a closed conformation
Summary
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. 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. To better understand the unique features of LigIV, we obtained two crystal structures of protein-DNA complexes in the ligation pathway—the lysyl-AMP intermediate in an open conformation, and the DNA–adenylate in a closed conformation Guided by these structures, we engineered several active site and surface mutants to probe their roles in the multistep catalytic cycle. Results from these studies provide insights into how hypomorphic mutations fundamentally alter enzymatic structure–function relationships, and establish a molecular basis for LIG4 Syndrome
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