The Saccharomyces cerevisiae ETH1 Gene, an Inducible Homolog of Exonuclease III That Provides Resistance to DNA-Damaging Agents and Limits Spontaneous Mutagenesis

Abstract

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1⁺ [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were ∼3-fold more sensitive to MMS and ∼10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were ∼15-fold more sensitive to MMS and ∼2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.

ACKNOWLEDGMENTS

I thank Bernard Weiss for providing bacterial strains for genetic complementation experiments, David M. Wilson III for construction of pKENAPE, and Yuji Masuda, Yong-jie Xu, Elisabeth M. Bailey, Brian J. Glassner, Scott A. Jelinsky, Bruce Demple, and Donny Wong for discussions and critical reading of the manuscript. Special recognition and thanks are given to Scott A. Jelinsky for the initial observation that ETH1 mRNA is induced by exposure of yeast to MMS and to Bruce Demple for providing financial support and encouragement.

This work was supported by NIH grants GM40000, ES03926, and CA71993 to B. Demple. R.A.O.B. is a Charles A. King Trust Fellow and is partially funded by Fleet Investment Management, Trustee of the Charles A. King Trust.

ADDENDUM IN PROOF

Bernard A. Connolly and his group in the United Kingdom recently demonstrated the conversion of bovine pancreatic DNase I to a DNA repair AP endonuclease. The recombinant protein has 14 amino acids, of which 10 are amino acids of an α-helix in exonuclease III that is not present in the natural form of DNase I, inserted at a position on the surface of DNase I corresponding to the location of the extra α-helix in exonuclease III (S. Cal, K. L. Tan, A. McGregor, and B. A. Connolly, EMBO J. 17:7128–7138, 1998).