Abstract
NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.
Highlights
At least one NADϩ-dependent DNA ligase is found in every bacterial species [2]
Domain Ia of E. coli DNA Ligase Is Required for Reaction with NADϩ—Prior studies had shown that N-terminal deletions N⌬78 and N⌬38 of E. coli DNA ligase (EcoLigA), which eliminate all or part of domain Ia, resulted in complete loss of nick joining activity [14]
A deletion of the N-terminal 38 amino acids of domain Ia had no effect on the rate or yield of the isolated step 3 reaction, and the more extensive N⌬78 deletion had only a modest (2-fold) effect on the rate of approach to the end point (Fig. 4A). These experiments show that specific functional groups within domain Ia of E. coli DNA ligase are important for the reaction of ligase with NADϩ but are not required for catalysis when the AMP pocket of the nucleotidyl transferase domain is filled by the adenylated DNA intermediate
Summary
Ligase Mutants—Missense mutations of domain Ia of EcoLigA were introduced into the pET-EcoLig expression plasmid using the PCRbased two-stage overlap extension method as described previously [14]. Assay of Nick Joining—Reaction mixtures (20 l) containing 50 mM Tris-HCl (pH 7.5), 10 mM (NH4)2SO4, 5 mM DTT, 5 mM MgCl2, 20 M NADϩ, 1 pmol of 5Ј-32P-labeled nicked duplex DNA substrate (shown in Fig. 2C), and aliquots of serial 2-fold dilutions of wild-type or mutant ligases were incubated at 22 °C for 10 min. The products were resolved by denaturing PAGE, and the extents of ligation were determined by scanning the gel with a FUJIX PhosphorImager. The DNA-adenylate ligation reaction mixtures (20 l) contained 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 5 mM MgCl2, nicked DNA-adenylate substrate, and wild-type or mutant EcoLigA proteins as specified. Reaction mixtures containing (per 20 l) 200 fmol of nicked DNA-adenylate substrate, 2 pmol of ligase, and other components as specified above were incubated at 22 °C. Aliquots (20 l) were withdrawn at the times specified and quenched immediately with EDTA and formamide [12]
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