Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4

We report that the NAD-dependent Escherichia coil DNA ligase can support th e growth of Saccharomyces cerevisiae strains deleted singly for CDCS or dou bly for CDCS plus LIG4. Alanine-scanning mutagenesis of E. coli DNA ligase led to the identification of seven amino acids (Lys115, Asp117, Asp285, Lys 314, Cys408, Cys411 and Cys432) that are essential for nick-joining in vitr o and for in vivo complementation in yeast. The K314A mutation uniquely res ulted in accumulation of the DNA-adenylate intermediate. Alanine substituti ons at five other positions (Glu113, Tyr225, Gln318, Glu319 and Cys426) did not affect in vivo complementation and had either no effect or only a mode st effect on nick-joining in vitro. The E113A and Y225A mutations increased the apparent K-m for NAD (to 45 and 76 mu M, respectively) over that of th e wild-type E. coli ligase (3 mu M). These results are discussed in light o f available structural data on the adenylylation domains of ATP- and NAD-de pendent ligases, We observed that yeast cells containing only the 298-amino acid Chlorella virus DNA ligase (a 'minimal' eukaryotic ATP-dependent liga se 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 es sential for mitotic growth, but may facilitate DNA repair.