Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-A crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO4 nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a beta-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO4 termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria and essential for growth. Their distinctive substrate specificity and domain organization vis-a-vis human ATP-dependent ligases make them outstanding targets for anti-infective drug discovery. We report here the 2.3 A crystal structure of Escherichia coli LigA bound to an adenylylated nick, which captures LigA in a state poised for strand closure and reveals the basis for nick recognition. LigA envelopes the DNA within a protein clamp. Large protein domain movements and remodeling of the active site orchestrate progression through the three chemical steps of the ligation reaction. The structure inspires a strategy for inhibitor design.

The DNA ligase D (LigD) 3′-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3′ end-healing reactions at DNA double-strand breaks. Here we report the 1.9 Å crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded β barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis à vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.is the key agent of the bacterial nonhomologous end-joining (NHEJ) pathway of DNA doublestrand break (DSB) repair (1). LigD is a single polypeptide consisting of three autonomous catalytic domain modules: an ATP-dependent ligase (LIG), a polymerase (POL), and a 3′-phosphoesterase (PE). The POL domain incorporates dNMP/rNMPs at DSB ends and gaps prior to strand sealing by the LIG domain (2-6) and is responsible, in large part, for the mutagenic outcomes of bacterial NHEJ in vivo (7). The PE domain provides a 3′ end-healing function, whereby it cleans up "dirty" DSBs with 3′-phosphate ends (8). PE also trims short 3′-ribonucleotide tracts (produced by POL) to generate the 3′ monoribonucleotide ends that are the preferred substrates for sealing by bacterial NHEJ ligases (3,8). The biochemical properties and atomic structures of the LIG and POL domains highlighted their membership in the covalent nucleotidyltransferase and archaeal/ eukaryal primase-polymerase families respectively (5, 9, 10). By contrast, the PE domain appears to be sui generis.The properties of the PE domain elucidated initially for Pseudomonas LigD also apply to the PE modules of Agrobacterium and Mycobacterium LigD (8,11,12). Specifically, PE displays a distinctive manganese-dependent 3′-ribonuclease/3′-phosphatase activity, entailing two component steps: (i) the 3′-terminal nucleoside is removed to yield a primer strand with a ribonucleoside 3′-PO 4 terminus; (ii) the 3′-PO 4 is hydrolyzed to a 3′-OH (Fig. 1A). PE activity is acutely dependent on the presence and length of a 5′ single-strand tail on a duplex primer-template substrate, thus implicating PE in 3′ end repair at gaps or recessed DSBs. Structure probing of Pseudomonas PE in solution revealed an apparently disordered N-terminal 29-aa segment, punctuated by a cluster of trypsin-and chymotrypsin-sensitive sites (Fig. 1B), flanking a seemingly well folded (i.e., protease insensitive) C-terminal domain (13). Deletion of the protease-sensitive N-terminal peptide had no effect on the phosphodiesterase activity of PE, though monoesterase activity was reduced. Mutational analyses identified an ensemble of conserved side chain functional groups within the protease-resistant module that were essential for ph...

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All LigA enzymes have a modular structure in which a central ligase catalytic core, composed of a nucleotidyltransferase (NTase) domain (amino acids 70 -316 in E. coli LigA) and an oligonucleotide-binding (OB) domain (amino acids 317-404), is flanked by an N-terminal "Ia" domain (amino acids 1-69) and three C-terminal modules: a tetracysteine zinc finger domain (amino acids 405-432), a helix-hairpin-helix (HhH) domain (amino acids 433-586), and a BRCA1-like C-terminal (BRCT) domain (amino acids 587-671) (4 -8). Each step of the ligation pathway depends upon a different subset of these modules.Domain Ia is unique to NAD ϩ -dependent ligases and is the determinant of NAD ϩ specificity (9 -11). During the ligase adenylylation reaction, the Ia domain closes over the NAD ϩ nucleotide bound by the NTase domain, grabs the NMN moiety of NAD ϩ via multiple contacts to essential amino acids in domain Ia, and thereby orients the NMN leaving group apical to the attacking lysine nucleophile (Lys 115 in E. coli LigA) (6, 10). Recognition of the AMP moiety of NAD ϩ and the catalysis of nucleotidyl transfer chemistry is accomplished by a constellation of essential amino acid side chains within the NTase domain that contact the adenine base, the ribose sugar, or the ␣-phosphate of the adenylate (5,6,8,12,13) (Fig. 1). Most of the essential NTase residues are located within a set of five peptide motifs that define a covalent nucleotidyltransferase superfamily, which includes ATP-dependent DNA ligases, ATP-dependent RNA ligases, and GTP-dependent mRNA capping enzymes (14). An N-terminal fragment of LigA, comprising just the Ia and NTase domains, is competent to catalyze ligase adenylylation (9,(15)(16)(17)(18), but it is unable to perform the second and third steps of the pathway. Deleting only the Ia domain from NAD ϩ -dependent ligases abolishes ligase adenylylation without affecting phosphodiester formation at a preadenylylated nick (step 3) (9 -11). These results, and others (9,15,16,18,19), implicate the C-terminal domains of NAD ϩ -dependent ligases in recognition of the DNA substrate.The crystal structure of E. coli LigA bound to the nicked DNA-adenylate intermediate (8) revealed that LigA encircles the DNA helix as a C-shaped protein clamp (Fig. 1). The protein-DNA interface entails extensive DNA contacts by the NTase, OB, and HhH domains over a 19-bp segment of duplex DNA centered about the nick (Fig. 1). The NTase domain binds to the broken DNA strands at and flanking the nick, the OB domain contacts the continuous template strand surrounding the nick, and the HhH domain binds both strands across the minor groove at the periphery of the footprint. The zinc finger module bridges the OB and HhH domains, and it contributes only a single main-chain amide contact to the DNA backbone (8). Domain Ia makes no contacts to the DNA duplex, consist-