Participation of glutamic acid 23 of T4 endonuclease V in theβ-elimination reaction of an abasic site in a synthetic duplex DNA

Abstract

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3′-phosphate by β-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Nati. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523–526, 1992). The results showed that replacement of Glu23 with either glutamine or asparatic acid completely abolished the glycosylase activity. We describe the Investigation of the second reaction (apurinic/apyrimidinic endo-nuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with asparatic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anlon in position 23 may act as a general base in the β-elimination reaction of the endonuclease.