PURIFICATION, CHARACTERIZATION, GENE CLONING, AND EXPRESSION OF SACCHAROMYCES-CEREVISIAE REDOXYENDONUCLEASE, A HOMOLOG OF ESCHERICHIA-COLI ENDONUCLEASE-III

Saccharomyces cerevisiae redoxyendonuclease (Scr), a homolog of Escher ichia coli endonuclease III, was purified from yeast deficient in the major apurinic/apyrimidinic endonuclease, Apn1. Studies of this highly purified preparation of Scr have revealed a number of similarities be tween this protein and endonuclease III as well as provided further ev idence for a common mechanism of action for this class of DNA glycosyl ase/AP lyases. We have employed a sensitive and specific assay for Scr which utilizes oligonucleotide substrates containing a single 5,6-dih ydrouracil base lesion or an abasic site. These substrates were utiliz ed to investigate the mode of action of Scr on damaged DNA and to comp are the kinetic properties of the yeast enzyme with its E. coil counte rpart. Furthermore, we have identified two distinct genes, SCR1 and SC R2, which encode highly homologous proteins with similar activities in yeast. Analysis of the deduced amino acid sequences of SCR1 and SCR2 suggests that S. cerevisiae possesses two similar enzymes encoded on s eparate chromosomes: one which bears an Fe-S binding motif, while the other does not. The potential biological roles of these two forms of-S cr are discussed.