Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent ligase known; it has an intrinsic nick-sensing function and suffices for yeast cell growth. Here, we report the 2.0 A crystal structure of the covalent ligase-AMP reaction intermediate. The conformation of the adenosine nucleoside and contacts between the enzyme and the ribose sugar have undergone a significant change compared to complexes of T7 ligase with ATP or mRNA capping enzyme with GTP. The conformational switch allows the 3' OH of AMP to coordinate directly the 5' PO(4) of the nick. The structure explains why nick sensing is restricted to adenylated ligase and why the 5' phosphate is required for DNA binding. We identify a metal binding site on ligase-adenylate and propose a mechanism of nick recognition and catalysis supported by mutational data.

mRNA capping occurs by a series of three enzymatic reactions in which the 5Ј triphosphate terminus of the primary transcript is cleaved to a diphosphate by RNA triphosphatase, capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA (guanine 7) methyltransferase (1). In vivo, the capping reactions occur cotranscriptionally, i.e. the substrates for the capping enzymes are nascent RNA chains engaged within RNA polymerase II (pol II) 1 elongation complexes. There must exist a mechanism to target cap formation in vivo to transcripts made by pol II, because the capping enzymes have no inherent specificity for modifying pre-mRNAs in vitro. We and others (2-4) have suggested that targeting is achieved through direct physical interaction of one or more components of the capping apparatus with the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of pol II. This model is supported by the finding that recombinant yeast guanylyltransferase and methyltransferase proteins bind specifically and independently to the phosphorylated CTD in vitro (2). Are such direct proteinprotein interactions conserved in higher eukaryotes? Does the triphosphatase component of the capping apparatus also interact with the CTD?We know that the physical and functional organizations of the triphosphatase and guanylyltransferase components of the capping apparatus have diverged in fungi versus metazoans. The guanylyltransferases of Saccharomyces cerevisiae (Ceg1; 459 amino acids), Schizosaccharomyces pombe (Pce1; 402 amino acids), and Candida albicans (Cgt1; 449 amino acids) are monofunctional polypeptides that cap diphosphate-terminated RNAs (5-7). Transfer of GMP from GTP to the 5Ј diphosphate terminus of RNA occurs in a two-stage reaction involving a covalent enzyme-GMP intermediate (8). The GMP is linked to the enzyme through a phosphoamide (P-N) bond to the ⑀-amino group of a lysine residue within a conserved motif, KXDG, found in all known cellular and DNA virus-encoded capping enzymes (9). The fungal guanylyltransferases display ϳ38% amino acid sequence identity overall. They are also functionally homologous, insofar as PCE1 and CGT1 can complement lethal ceg1 mutations in S. cerevisiae (6, 7). The S. cerevisiae RNA triphosphatase is a 549-amino acid polypeptide encoded by the CET1 gene (10). The Ceg1 and Cet1 polypeptides interact in vivo and in vitro.Metazoan capping enzymes are bifunctional polypeptides with triphosphatase and guanylyltransferase activities (11,12). Yagi et al. (12) isolated triphosphatase and guanylyltransferase domain fragments of the Artemia salina capping enzyme by partial proteolysis with trypsin. However, it was not clear from this work whether the functional domains overlapped structurally. The first metazoan-capping enzyme gene was isolated recently from Caenorhabditis elegans (13,14). The 573-amino acid nematode protein consists of a carboxyl-terminal domain homologous to yeast Ceg1 and an amino-terminal domain that has strong similarity to the superfamil...

A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.

We report that the NAD-dependent Escherichia coli DNA ligase can support the growth of Saccharomyces cerevisiae strains deleted singly for CDC9 or doubly for CDC9 plus LIG4. Alanine-scanning mutagenesis of E.coli DNA ligase led to the identification of seven amino acids (Lys115, Asp117, Asp285, Lys314, Cys408, Cys411 and Cys432) that are essential for nick-joining in vitro and for in vivo complementation in yeast. The K314A mutation uniquely resulted in accumulation of the DNA-adenylate intermediate. Alanine substitutions at five other positions (Glu113, Tyr225, Gln318, Glu319 and Cys426) did not affect in vivo complementation and had either no effect or only a modest effect on nick-joining in vitro. The E113A and Y225A mutations increased the apparent K (m)for NAD (to 45 and 76 microM, respectively) over that of the wild-type E. coli ligase (3 microM). These results are discussed in light of available structural data on the adenylylation domains of ATP- and NAD-dependent ligases. We observed that yeast cells containing only the 298-amino acid Chlorella virus DNA ligase (a 'minimal' eukaryotic ATP-dependent ligase consisting only of the catalytic core domain) are relatively proficient in the repair of DNA damage induced by UV irradiation or treatment with MMS, whereas cells containing only E.coli ligase are defective in DNA repair. This suggests that the structural domains unique to yeast Cdc9p are not essential for mitotic growth, but may facilitate DNA repair.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.