Apn2 is a member of the Exo III/HAP1 family of proteins. (A) Alignment of human HAP1, S. cerevisiae Apn2, and E. coli Exo III (Xth) proteins. Identical and highly conserved residues are highlighted. Amino acid positions are indicated by numbers in parentheses. The conserved residues thought to be involved in catalytic activity (Glu-96, Asp-283, and His-309 in HAP1 protein) are indicated by arrows. The Cys-65 residue in HAP1 essential for the redox activity is circled and indicated by an asterisk. (B) Schematic alignment of Exo III-type nucleases from various organisms. Boxes represent primary amino acid sequence. Narrow regions indicate unique sequences, whereas larger, shaded, or stippled regions indicate regions of homology among the proteins. The different shades within the boxes indicate independent domains; spaces indicate gaps that were introduced to optimize alignment of the proteins. Protein lengths are indicated by numbers at right. Positions of the highly conserved amino acid sequences QE(T/L/I)K, R(L/I)D, and SDH(C/A)P are indicated. Positions of the redox cysteine residue in human HAP1, and of cysteine residues present at the corresponding position in Drosophila RRP and Arabidopsis thaliana ARP proteins are indicated by the letter C within the alignments.

Enhanced MMS sensitivity and MMS mutagenesis in the apn1Δ apn2Δ strain. (A) MMS sensitivity of various yeast strains. Cells grown overnight in YPD medium were treated with MMS at the concentrations indicated for a 20-min period. Appropriate dilutions were spread onto YPD plates. Each curve represents the average of two to three experiments for each strain. (•) EMY74.7, wild type (APN1 APN2); (□) YRP190, apn1Δ; (▵) YRP263, apn2Δ; (○) YRP269, apn1Δ apn2Δ. (B) MMS-induced mutations at the CAN1 locus. Cells grown overnight in YPD medium were treated with MMS at the concentrations indicated for a 20-min period. Appropriate dilutions were spread onto YPD plates for viability determinations and onto synthetic complete medium lacking arginine and containing canavanine for the determination of CAN1^S to can1^r mutagenesis. Each curve represents the average of two to three experiments for each strain. (•) EMY74.7, wild type (APN1 APN2); (□) YRP190, apn1Δ; (▵) YRP263, apn2Δ; (○) YRP269, apn1Δ apn2Δ.

Alkaline sucrose gradient analysis of DNA from cells treated with 0.1% MMS for 20 min. (A) YRP276, wild type (APN1 APN2). (B) YRP210, apn1Δ. (C) YRP291, apn2Δ. (D) YRP292, apn1Δ apn2Δ. (○) Untreated cells; (•) cells treated with MMS for 20 min; (▴) cells treated with MMS for 20 min, and then given a 1-hr repair period; (▵) cells treated with MMS for 20 min, and then given a 2-hr repair period.

Requirement of REV genes for MMS-induced mutagenesis in the apn1Δ apn2Δ strains. Methods are as described in the legend to Fig. 2. (A) MMS sensitivity of yeast strains. (•) EMY74.7, wild type (APN1 APN2); (○) YRP269, apn1Δ apn2Δ; (▵) YREV1.15, rev1Δ; (█) YREV3.15, rev3Δ; (▾) YREV7.1, rev7Δ; (▴) YREV1.13, apn1Δ apn2Δ rev1Δ; (□) YREV3.40, apn1Δ apn2Δ rev3Δ; (▿) YREV7.4, apn1Δ apn2Δ rev7Δ. (B) MMS-induced mutations of CAN1^S to can1^r. (○) YRP269, apn1Δ apn2Δ; (▴) YREV1.13, apn1Δ apn2Δ rev1Δ; (□) YREV3.40, apn1Δ apn2Δ rev3Δ; (▿) YREV7.4, apn1Δ apn2Δ rev7Δ.