Truncation of Amino-terminal Tail Stimulates Activity of Human Endonuclease III (hNTH1)

Abstract

The human base excision repair enzyme hNTH1, a homologue of Escherichia coli endonuclease III (Nth), is a 36 kDa DNA glycosylase with associated abasic (AP) lyase activity. It has significant sequence homology with Nth in its DNA-binding motifs and catalytic residues but possesses a unique amino (N)-terminal tail (residues 1–95). We investigated the structure and function of this tail. Controlled proteolysis cleaved hNTH1 into discrete fragments to generate a 25 kDA core domain lacking the N-terminal 98 residues. Surprisingly, recombinant hNTH1 lacking 55, 72 or 80 residues from the N terminus had four- to fivefold higher activities than the full-length enzyme. Kinetic analysis at transition states revealed that release of the final product, an AP site with a 3′-nick, is the rate-limiting step in the multi-step reaction mediated by hNTH1. The N-terminal tail regulates its overall catalytic turnover by reducing this product release rate by five- to sevenfold without affecting either the glycosylase or AP lyase activities, or the steady-state equilibrium concentration of Schiff base intermediate, the covalent complex of hNTH1 and AP-site DNA formed after the base is excised. The inhibitory role of the N-terminal tail in catalytic turnover explains the low activity of hNTH1 compared to that of its E. coli homologue.