Supplementary Figure 2: Patch test reveals that inactivation of both the MIP and SHIP box motifs in Exo1 cause a defect in Exo1-dependent MMR but not in the ability of Exo1 to suppress the synthetic lethality of exo1Δ and rad27Δ mutations.

a, Low-copy-number ARS-CEN plasmids without an insert or bearing different EXO1 constructs were tested for their ability to suppress the mutator phenotype of the exo1Δ pol30^K217E double mutant (RDKY8077) or the exo1Δ pms1^A99V double mutant (RDKY4192). Empty vector and vector bearing the nuclease-dead exo1^D173A allele were unable to suppress the mutator phenotype, while the wild-type EXO1 substantially suppressed the increased mutation rate of the double mutant. An allele of EXO1 containing mutations that disrupted the MIP box (exo1^F447A,F448A) and deleted the region including both SHIP boxes (exo1^Δ571–702) was unable to suppress the mutator phenotype, equivalent to the empty vector or the nuclease-dead exo1^D173A allele. All experiments were independently repeated a minimum of two times. b, An S. cerevisiae rad27Δ exo1Δ double-mutant strain containing a wild-type EXO1-bearing URA3 plasmid was transformed with TRP1 plasmids either without EXO1 or with various EXO1 mutations. The transformed strains were then plated either on YPD or CSM medium containing 5FOA to select for cells that lost the complementing EXO1-bearing URA3 plasmid. Neither the empty vector nor the nuclease-dead exo1^D173A allele could suppress the synthetic lethality of the rad27Δ exo1Δ▯double mutations and grow on 5FOA-containing medium. In contrast, alleles of EXO1 containing mutations that disrupted the MIP box (exo1^F447A,F448A) or deleted the region including both SHIP boxes (exo1^Δ571–702) or lacked both functional MIP or SHIP boxes (exo1^{F447A,F448A,Δ571–702}) would support growth on 5FOA-containing medium. Thus, defects in the MIP and SHIP boxes that cause MMR defects do not disrupt the functions of Exo1 required for survival of rad27Δ strains. All experiments were independently repeated a minimum of two times.