Dynamic mechanism of nick recognition by DNA ligase

Abstract

DNA ligases are the enzymes responsible for the repair of single-stranded and double-stranded nicks in dsDNA. DNA ligases are structurally similar, possibly sharing a common molecular mechanism of nick recognition and ligation catalysis. This mechanism remains unclear, in part because the structure of ligase in complex with dsDNA has yet to be solved. DNA ligases share common structural elements with DNA polymerases, which have been cocrystallized with dsDNA. Based on the observed DNA polymerase-dsDNA interactions, we propose a mechanism for recognition of a single-stranded nick by DNA ligase. According to this mechanism, ligase induces a B-to-A DNA helix transition of the enzyme-bound dsDNA motif, which results in DNA contraction, bending and unwinding. For non-nicked dsDNA, this transition is reversible, leading to dissociation of the enzyme. For a nicked dsDNA substrate, the contraction of the enzyme-bound DNA motif (a) triggers an opened-closed conformational change of the enzyme, and (b) forces the motif to accommodate the strained A/B-form hybrid conformation, in which the nicked strand tends to retain a B-type helix, while the non-nicked strand tends to form a shortened A-type helix. We propose that this conformation is the catalytically competent transition state, which leads to the formation of the DNA-AMP intermediate and to the subsequent sealing of the nick.Keywords: DNA ligase; nick recognition; A-form DNA; A/ B-form DNA hybrid; protein-DNA interactions; B-A DNA helix transition.DNA ligases are the enzymes that catalyze the joining of single-and double-stranded nicks in dsDNA [1]. These enzymes play a pivotal role in replication, sealing the nicks in the lagging DNA strand [2][3][4][5]. They also participate in DNA excision [6][7][8], double-strand break repair [9][10][11][12] and take part in DNA recombination [10,[13][14][15]. The mechanism of enzyme catalysis (Scheme 1) includes three main steps: (1) covalent binding of the nucleoside monophosphate, AMP or GMP, via the e-amino lysyl phosphoramidate bond, (2) transfer of the nucleotidyl moiety onto the 5¢-phosphate end of the nick, forming an inverted pyrophosphate bridging structure, A(G)ppN and (3) formation of the phosphodiester bond between the 3¢-OH and the 5¢-phosphate ends of the nick, releasing the nucleotide. Scheme 1. Mechanism of the ATP-dependent end-joining activity of T4 DNA ligase. nds-DNA, dsDNA containing a 5¢-phosphorylated nick. n-MgAMP-dsDNA, nicked dsDNA adenylylated at the 5¢-phosphate of the nick.

Effects of Deletion and Site-Directed Mutations on Ligation Steps of NAD+-Dependent DNA Ligase: A Biochemical Analysis of BRCA1 C-Terminal Domain

“…Recent conformational analyses of E. coli and Thermus scotoductus NAD + ligases have led to a model in which the adenylation-dependent open-close conformational change induces the ligase to a catalytically activated state (52,53). Similar open-close models are proposed for T7 ligase, T4 ATP ligase, and mRNA capping enzyme (17,(54)(55)(56).…”

Construction, Stability, and Activity of Multivalent Circular Anticoagulant Aptamers

“…Enzymatic Ligation-Although the structure of T4 DNA ligase has not been solved, amino acid sequence similarity between many ligases is high (47), and the reported three-dimensional structures of ligases show highly conserved structural domains (48). Footprinting and modeling studies of T7 DNA ligase bound to nicked duplexes show contact of 7-9 nucleotides on the 5Ј side of the nick and 3-5 nucleotides on the 3Ј side (47).…”

Probing transient protein-mediated DNA linkages using nanoconfinement

“…ATP is required for specific binding of ligase to both DNA strands, while Mg 2þ is required for catalytic action. 46,47 At high T4 DNA ligase concentration, non-specific binding to dsDNA was observed. Furthermore, it was proposed that the apparent persistence length of DNA drops in presence of a high concentration T4 DNA ligase, 48,49 which is likely due to ligase induced DNA kinking and local base-pair opening in transient DNA-ligase complexes.…”