Conserved Residues in Domain Ia Are Required for the Reaction of Escherichia coli DNA Ligase with NAD+

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 ligaseadenylate 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. DNA ligases are grouped into two families, ATP-dependent ligases and NADϩ -dependent ligases, according to the cofactor required for ligase-adenylate formation (1). Both types of DNA ligases catalyze the sealing of 5Ј-phosphate and 3Ј-hydroxyl termini at nicks in duplex DNA by means of three sequential nucleotidyl transfer reactions. In the first step, attack on the ␣ phosphorus of ATP or NAD ϩ by ligase results in release of pyrophosphate or nicotinamide mononucleotide (NMN) 1 and formation of a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide bond to lysine. In the second step, the AMP is transferred to the 5Ј end of the 5Ј-phosphate-terminated DNA strand to form DNA-adenylate (AppDNA). In the third step, ligase catalyzes attack by the 3Ј-OH of the nick on DNA-adenylate to join the two polynucleotides and release AMP.

Characterization of a temperature-sensitive DNA ligase from Escherichia coli

“…Although the relationship of the amino acid mutation to enzymic activity or structure has not been directly demonstrated at this time, the molecular model suggests that the alteration of amino acid 15 from Leu to Phe may alter the packing involving a-helix A, which is close to the N-terminus. This bi-helix is implicated in the binding of NAD + to LigA (Georlette et al, 2003;Sriskanda & Shuman, 2002), supporting our proposal that the temperature sensitivity of LigA251 is a consequence of factors altering the rate of adenylation of the protein.…”

“…This rate is comparable to that reported for other recombinant NAD + -DNA ligases (e.g. see Kaczmarek et al, 2001;Sriskanda & Shuman, 2002;Tong et al, 2000). At 20 u C, the rate of DNA ligation by LigA251 was about 20-fold lower than that of LigA.…”

“…In the NAD + -DNA ligases, this amino acid resides within the bi-helix that is close to the N-terminus. Since this region of LigA is important for binding of NAD + (Sriskanda & Shuman, 2002), the ts mutation in E. coli GR501 is likely to affect binding of NAD + and the subsequent adenylation of LigA.…”

“…Thus, NAD + -DNA ligases have been suggested as possible targets for broad-spectrum antibacterial compounds (Georlette et al, 2003;Gong et al, 2004;Kaczmarek et al, 2001;Lee et al, 2000;Singleton et al, 1999;Sriskanda & Shuman, 2002). If substantial progress is to be made in the development of inhibitors that are specific to NAD + -DNA ligases, it is vital that in vivo models are utilized to test the efficacy of such compounds.…”

Cofactor Binding Modulates the Conformational Stabilities and Unfolding Patterns of NAD+-dependent DNA Ligases from Escherichia coli and Thermus scotoductus

“…Such a conformational change is likely to prevent the formation of non-productive ligase-DNA complexes in the cell. Additional information has also been drawn from the investigation of the role of Escherichia coli domain Ia (12). This domain, unique to NAD ϩ -dependent ligases, is essential for the reaction with NAD ϩ .…”