FUNCTIONAL EXPRESSION OF ESCHERICHIA-COLI ENDONUCLEASE-IV IN APURINICENDONUCLEASE-DEFICIENT YEAST

Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV are homologous enzymes that initiate the repair of abasic (AP) sites or o xidative DNA strand breaks. Yeast lacking Apn1 (apn1(-)) are hypersens itive to simple alkylating agents (which produce many AP sites) and to oxidants and display an elevated spontaneous mutation rate due to end ogenous damages. We explored whether the prokaryotic repair enzyme cou ld substitute for its yeast counterpart. Plasmid constructs were gener ated that expressed endonuclease IV at 1/20 to 10-fold the AP endonucl ease activity of wild-type yeast; some of these plasmids expressed hyb rid forms of endonuclease IV equipped with the C-terminal nuclear loca lization signal of Apn1. Although hybrid endonuclease IV-Apn1 (but not native endonuclease IV) was selectively localized to the yeast nucleu s, expression of this chimeric protein at 25% of the normal Apn1 level did not restore alkylation or oxidant resistance to apn1(-) yeast, bu t it did partially counteract the mutator phenotype of apn1(-) yeast. Expression of either the hybrid protein or native endonuclease IV at s imilar to 10 times wild-type Apn1 levels restored wild-type resistance to methyl methanesulfonate and near-wild-type H2O2 resistance. High l evel expression of native endonuclease IV also restored the normal spo ntaneous mutation rate to apn1(-) yeast. These data place limits on th e amounts of AP endonuclease activity necessary for repair of DNA dama ges caused by both endogenous and environmental agents and point to a direct role of spontaneous AP sites as potentially mutagenic lesions.