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. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 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.

FIG. 1

(A) Protein alignment for selected AP endonucleases of the exonuclease III family. Gray shading indicates sequences that do not have sequence homology to exonuclease III. The percent amino acid identity relative to human AP endonuclease (Ape1) is given for the recombination protein (ARP) of Arabidopsis, the recombination repair protein (RRP1) of Drosophila melanogaster, exonuclease A (ExoA) of Streptococcus pneumoniae, exonuclease III (Xth) of Escherichia coli, the exonuclease III homolog (ScEth1) of Saccharomyces cerevisiae, and the exonuclease III homolog (SpEth1) of Schizosaccharomyces pombe. The vertical bars in the AP endonuclease domain (open box) show highly conserved residues encompassing the proposed catalytic amino acids; other conserved short regions exist but are not indicated (see panel B for details). (B) Partial amino acid sequence alignment of nine selected exonuclease III family members over the DNA repair domains of these proteins. Asterisks indicate proposed catalytic amino acids (, –31). Dots indicate gaps in the sequences. A black background indicates amino acid identity, while gray shading indicates amino acid similarity. Pileup was used to align the sequences (15).

FIG. 1

(A) Protein alignment for selected AP endonucleases of the exonuclease III family. Gray shading indicates sequences that do not have sequence homology to exonuclease III. The percent amino acid identity relative to human AP endonuclease (Ape1) is given for the recombination protein (ARP) of Arabidopsis, the recombination repair protein (RRP1) of Drosophila melanogaster, exonuclease A (ExoA) of Streptococcus pneumoniae, exonuclease III (Xth) of Escherichia coli, the exonuclease III homolog (ScEth1) of Saccharomyces cerevisiae, and the exonuclease III homolog (SpEth1) of Schizosaccharomyces pombe. The vertical bars in the AP endonuclease domain (open box) show highly conserved residues encompassing the proposed catalytic amino acids; other conserved short regions exist but are not indicated (see panel B for details). (B) Partial amino acid sequence alignment of nine selected exonuclease III family members over the DNA repair domains of these proteins. Asterisks indicate proposed catalytic amino acids (, –31). Dots indicate gaps in the sequences. A black background indicates amino acid identity, while gray shading indicates amino acid similarity. Pileup was used to align the sequences (15).

FIG. 2

MMS, hydrogen peroxide, and phleomycin D1 toxicity. Strains are as follows: □, MKP-o; ◊, DRY373; ○, RBY31; ▵, RBY71. The genotypes of these strains are given in the figure. All data are plotted as means and standard deviations from at least three independent experiments. For the phleomycin data, the standard deviations are smaller than the size of the data points. For the limit of detection for this assay, more than 10 colonies had to survive from 2 × 10⁶ cells plated.

FIG. 3

Replacement of ETH1. MKP-o (wild type), DRY373 (apn1), RBY31 (eth1), and RBY71 (apn1 eth1) contain a yeast expression vector, pYES2 (top four strains in each panel), or the ETH1 expression vector, pETH1 (bottom four strains in each panel). The growth medium used for the gradient plate is CM−ura+gal (selection for the plasmid and inducing conditions [A to D]) or CM−ura+glu (selection for the plasmid and noninducing conditions [E to H]). The bottom layer does not contain MMS (A and E) or contains 0.00313% MMS (B and F), 0.00625% MMS (C and G), or 0.025% MMS (D and H).

FIG. 4

Spontaneous reversion rates for lys2-1 and ade2-1. The relative increase in mutation rate is plotted with respect to that of the wild-type strain MKP-o, which is defined as 1. Data are means and standard deviations from three independent experiments.

FIG. 5

Complementation of E. coli BW287. Three plasmids were transformed into BW287: pKEN2 (vector control), pKENETH (ETH1 expression), and pKENAPE (human AP endonuclease protein expression). The plates were incubated at the indicated temperatures for 24 h.

FIG. 6

Northern blot analysis of ETH1. Total RNA was harvested from DBY747, and 20 μg was loaded into each lane. Following electrophoresis and transfer to a positively charged nylon membrane (Boehringer Mannheim), the RNA was hybridized to a radioactive internal BglII fragment of ETH1. (A) 28S and 18S rRNA revealed by ethidium bromide staining. (B) Hybridization intensity of the radiolabeled probe to ETH1 mRNA revealed by phosphorimager analysis (Bio-Rad) of RNA from the gel shown in panel A. (C) Fold induction shown from Northern analysis of three independent measurements from two experiments. To correct for RNA-loading differences, the phosphorimager data in panel B are normalized to the quantified intensities of the 28S and 18S rRNA shown in panel A. Data are presented as means and standard deviations (n = 3).