The HAP1 protein stimulates the turnover of human mismatch-specific thymine-DNA-glycosylase to process 3,N-4-ethenocytosine residues

When present in DNA, 3,N-4-ethenocytosine (epsilonC) residues are potential ly mutagenic and carcinogenic in vivo. The enzymatic activity responsible f or the repair of the epsilonC residues in human cells is the hTDG protein, the human thymine-DNA-glycosylase that removes thymine in a T/G base pair [ Proc. Natl. Acad. Sci., U.S.A., 95 (1998) 8508]. One of the distinctive pro perties of the hTDG protein is that it remains tightly bound to the AP-site resulting from its glycosylase activity. In this paper we report that the human AP endonuclease, the HAP1 (Ape1, APEX Ref-1) protein, stimulates the processing of epsilonC residues by the hTDG protein in vitro, in a dose-dep endent manner. This property of HAP1 protein is specific since E.coli Fpg, Nfo and Nth proteins, all endowed with an AP nicking activity, do not show similar features. The results suggest that the HAP1 protein displaces the h TDG protein bound to the AP-site and therefore increases the turnover of th e hTDG protein. However, using a variety of techniques including gel retard ation assay, surface plasmon resonance and two-hybrid system, it was not po ssible to detect evidence for a complex including the substrate, the hTDG a nd HAP1 proteins. (C) 2001 Elsevier Science B.V. All rights reserved.