SACCHAROMYCES-CEREVISIAE POSSESSES 2 FUNCTIONAL HOMOLOGS OF ESCHERICHIA-COLI ENDONUCLEASE-III

We previously identified two distinct genes of Saccharomyces cerevisia e redoxyendonuclease (SCR1 and SCR2) which possess a high degree of se quence similarity to Escherichia coli endonuclease III [Augeri, L., Le e, Y. M., Barton, A. B., and Doetsch, P. W. (1997) Biochemistry 36, 72 1-729]. The proteins encoded by SCR1 and SCR2 were overexpressed in E. call and purified to apparent homogeneity. Both proteins recognized a nd cleaved DNA substrates containing dihydrouracil, 2,6-diamino-4-hydr oxy-5N-methylformamidopyrimidine 5N-methylformamidopyrimidine (FaPy-7- MeGua), and abasic;sites but not DNA substrates containing uracil or 8 -oxoguanine. Purified Scr2, but not Scr1, possesses spectral propertie s which indicate the presence of an iron-sulfur center. Kinetic parame ters for Scr1 and Scr2 were determined by using an oligonucleotide con taining a single dihydrouracil. Analysis of the deduced amino acid seq uences of Scr1 and Scr2 suggests that Scr2 bears an iron-sulfur motif, while Scr1 does not have this motif. However, Scr1 has a long, positi vely charged N-terminus that could be a mitochondrial transit sequence . Targeted gene disruption of SCR1 and SCR2 produced a double mutant t hat had no detectable enzymatic activity against the dihydrouracil-con taining substrate. Northern blot analysis showed that SCR1 was induced by menadione, but SCR2 was not. These results indicate that although Scr1 and Scr2 are both functional homologues of E. coli endonuclease I II, they differ from each other with respect to their amino acid seque nces and inducibility by DNA damaging agents, suggesting that their pr ecise biological roles may be different.