Purification, characterization, gene cloning, and expression of Saccharomyces cerevisiae redoxyendonuclease, a homolog of Escherichia coli endonuclease III.

Saccharomyces cerevisiae redoxyendonuclease (Scr), a homolog of Escherichia coli endonuclease III, was purified from yeast deficient in the major apurinic/apyrimidinic endonuclease, Apnl. Studies of this highly purified preparation of Scr have revealed a number of similarities between this protein and endonuclease III as well as provided further evidence for a common mechanism of action for this class of DNA glycosylase/AP lyases. We have employed a sensitive and specific assay for Scr which utilizes oligonucleotide substrates containing a single 5,6-dihydrouracil base lesion or an abasic site. These substrates were utilized to investigate the mode of action of Scr on damaged DNA and to compare the kinetic properties of the yeast enzyme with its E. coli counterpart. Furthermore, we have identified two distinct genes, SCR1 and SCR2, 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 separate chromosomes: one which bears an Fe-S binding motif, while the other does not. The potential biological roles of these two forms of Scr are discussed.

A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities.

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.