INTRACELLULAR-LOCALIZATION OF THE APN1 DNA-REPAIR ENZYME OF SACCHAROMYCES-CEREVISIAE - NUCLEAR TRANSPORT SIGNALS AND BIOLOGICAL ROLE

The Apn1 DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages. Apn1 is homologous to the repair end onuclease IV of Escherichia coli, but the yeast protein is approximate ly 80 residues longer at the C terminus. The Apn1 C terminus is rich i n basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. We show here by indirect immunofluorescence that Apn1 is localized to the yeast nuc leus. Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus. Elimination of just the last 12 residues f rom Apn1 (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme. Greater truncation of Apn1 pro duced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability. Both Apn355 and Apn334 failed to concentrat e in the yeast nucleus and remained in the cytoplasm. These delocalize d derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a DELTAapn1 strain. Apn355 and Apn334 complem ented repair-deficient E. coli as effectively, as did wild-type Apn1. Resistance to these DNA-damaging agents in yeast was restored if Apn35 5 and Apn334 (but not Apn315 or Apn293) were overproduced approximatel y 20-fold, which suggests either weak active transport or passive diff usion of these derivatives into the nucleus. Replacement of the C-term inal 12 residues of Apn1 with the nuclear targeting sequence of SV40 T -antigen did not restore effective function or nuclear localization in yeast.