A second NAD+-dependent DNA ligase (LigB) in Escherichia coli

Abstract

Escherichia coli DNA ligase (LigA) is the prototype of the NAD+-dependent class of DNA ligases found in all bacteria. Here we report the characterization of E. coli LigB, a second NAD+-dependent DNA ligase identified by virtue of its sequence similarity to LigA. LigB differs from LigA in that it lacks the BRCA1 C-terminus domain (BRCT) and two of the four Zn-binding cysteines that are present in LigA and all other bacterial NAD+ ligases. We found that recombinant LigB catalyzed strand joining on a singly-nicked DNA in the presence of a divalent cation and NAD+, and that LigB reacted with NAD+ to form a covalent ligase-adenylate intermediate. Alanine substitution for the motif I lysine (126KxDG) abolished nick joining and ligase-adenylate formation by LigB, thus confirming that the ligase and adenylyltransferase activities are intrinsic to the LigB protein.