A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

Abstract

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.

Figure 1

EXO1 acts in the MMS2 error-free branch of PRR in response to MMS independent of MMR. Overnight saturated cultures were serially diluted (1:5), spotted on YPD plates and YPD plates with increasing concentrations of MMS using a 48-prong replicator and then incubated at 30 °C for 2-4 days. The exo1Δ mutation defines an MMS tolerance pathway that functions in the (A) RAD6/RAD18 PRR pathway, but is redundant with the REV3 error-prone branch and the RAD9 checkpoint branch as the double (B) exo1Δ rad9Δ and (C) exo1Δ rev3Δ mutants are synergistically more sensitive than the single mutants alone. In contrast, (D) EXO1 is hypostatic to MMS2 and other components of the error-free branch of the PRR, but EXO1 did not interact with pathways competing with PRR as defined by MGS1 and RAD30. This EXO1 DNA damage tolerance pathway is independent of MMR as there was no genetic interaction when (C) msh2Δ or (Figure S1B) pms1Δ mutants were examined.

Figure 1

EXO1 acts in the MMS2 error-free branch of PRR in response to MMS independent of MMR. Overnight saturated cultures were serially diluted (1:5), spotted on YPD plates and YPD plates with increasing concentrations of MMS using a 48-prong replicator and then incubated at 30 °C for 2-4 days. The exo1Δ mutation defines an MMS tolerance pathway that functions in the (A) RAD6/RAD18 PRR pathway, but is redundant with the REV3 error-prone branch and the RAD9 checkpoint branch as the double (B) exo1Δ rad9Δ and (C) exo1Δ rev3Δ mutants are synergistically more sensitive than the single mutants alone. In contrast, (D) EXO1 is hypostatic to MMS2 and other components of the error-free branch of the PRR, but EXO1 did not interact with pathways competing with PRR as defined by MGS1 and RAD30. This EXO1 DNA damage tolerance pathway is independent of MMR as there was no genetic interaction when (C) msh2Δ or (Figure S1B) pms1Δ mutants were examined.

Figure 1

EXO1 acts in the MMS2 error-free branch of PRR in response to MMS independent of MMR. Overnight saturated cultures were serially diluted (1:5), spotted on YPD plates and YPD plates with increasing concentrations of MMS using a 48-prong replicator and then incubated at 30 °C for 2-4 days. The exo1Δ mutation defines an MMS tolerance pathway that functions in the (A) RAD6/RAD18 PRR pathway, but is redundant with the REV3 error-prone branch and the RAD9 checkpoint branch as the double (B) exo1Δ rad9Δ and (C) exo1Δ rev3Δ mutants are synergistically more sensitive than the single mutants alone. In contrast, (D) EXO1 is hypostatic to MMS2 and other components of the error-free branch of the PRR, but EXO1 did not interact with pathways competing with PRR as defined by MGS1 and RAD30. This EXO1 DNA damage tolerance pathway is independent of MMR as there was no genetic interaction when (C) msh2Δ or (Figure S1B) pms1Δ mutants were examined.

Figure 1

EXO1 acts in the MMS2 error-free branch of PRR in response to MMS independent of MMR. Overnight saturated cultures were serially diluted (1:5), spotted on YPD plates and YPD plates with increasing concentrations of MMS using a 48-prong replicator and then incubated at 30 °C for 2-4 days. The exo1Δ mutation defines an MMS tolerance pathway that functions in the (A) RAD6/RAD18 PRR pathway, but is redundant with the REV3 error-prone branch and the RAD9 checkpoint branch as the double (B) exo1Δ rad9Δ and (C) exo1Δ rev3Δ mutants are synergistically more sensitive than the single mutants alone. In contrast, (D) EXO1 is hypostatic to MMS2 and other components of the error-free branch of the PRR, but EXO1 did not interact with pathways competing with PRR as defined by MGS1 and RAD30. This EXO1 DNA damage tolerance pathway is independent of MMR as there was no genetic interaction when (C) msh2Δ or (Figure S1B) pms1Δ mutants were examined.

Figure 2

Defining separate Exo1p PRR and MMR domains. (A) Schematic and summary of phenotypes for Exo1p deletion mutants tested by MMS sensitivity (PRR) and for MMR mutator as shown in Figure 2B and Table 2, respectively. Exo1p with functional domains and motifs as indicated by respective hatched boxes. Site-specific mutations experimentally examined are highlighted by asterisks as detailed in the text. This analysis defines Exo1p deletion mutant #4 (1-438 aa) as a separation of function mutant. (B) Complementation of MMS tolerance in an exo1Δ rev3Δ strain with the listed Exo1p deletion mutant constructs suggests that residues 1-438 are necessary for functional PRR. MMS sensitivity was performed as described previously in Figure 1.

Figure 2

Defining separate Exo1p PRR and MMR domains. (A) Schematic and summary of phenotypes for Exo1p deletion mutants tested by MMS sensitivity (PRR) and for MMR mutator as shown in Figure 2B and Table 2, respectively. Exo1p with functional domains and motifs as indicated by respective hatched boxes. Site-specific mutations experimentally examined are highlighted by asterisks as detailed in the text. This analysis defines Exo1p deletion mutant #4 (1-438 aa) as a separation of function mutant. (B) Complementation of MMS tolerance in an exo1Δ rev3Δ strain with the listed Exo1p deletion mutant constructs suggests that residues 1-438 are necessary for functional PRR. MMS sensitivity was performed as described previously in Figure 1.

Figure 3

The FF->AA mutation prevents Exo1p from interacting with Mlh1p, but not Msh2p by yeast two-hybrid. Strains with designated bait-prey sets were grown in nonselective media (-TRP -URA) to saturation, serially diluted (1:5), spotted on the indicated plates using a 48-prong replicator and then incubated at 30 °C for 3 days. Strains were assayed for β-gal activity by lift assays as described in the Materials and Methods. Growth on –HIS plates and blue color development on the β -gal assay indicates interaction between the bait and prey fusion proteins. (A) Mlh1p-LexAp bait and Exo1p-Gad4p prey combinations demonstrate that the exo1-FF447AA mutation prevents Exo1p-Gad4p interaction with Mlh1p-LexAp. Lanes: 1, pBTM (empty bait control) + pGAD (empty prey control); 2, pBTM-MLH1 + pGAD; 3, pBTM-MLH1 + pGAD-EXO1; 4, pBTM-MLH1 + pGAD-exo1-FF447AA # 1; 5, pBTM-MLH1 + pGAD-exo1-FF447AA # 5; 6, pBTM-MLH1 + pGAD-exo1-FF447AA # 6; and 7, pBTM-MLH1 + pGAD-exo1-FF447AA # 10. (B) Msh2p-LexAp bait and Exo1p-Gad4p prey combinations demonstrate that the exo1-FF447AA mutation does not prevent Exo1p-Gad4p interaction with Msh2p-LexAp. Lanes: 1, pBTM (control) + pGAD (control); 2, pBTM-MSH2 + pGAD; 3, pBTM- MSH2 + pGAD-EXO1; 4, pBTM- MSH2 + pGAD-exo1-FF447AA # 1; 5, pBTM- MSH2 + pGAD-exo1-FF447AA # 5; 6, pBTM- MSH2 + pGAD-exo1-FF447AA # 6; and 7, pBTM- MSH2 + pGAD-exo1-FF447AA # 10. Clones pGAD-exo1-FF447AA #1, 5, 6 & 10 represent four independently generated “prey” constructs.

Figure 4

The exo1-FF447AA mutant is functional for PRR. Overnight saturated cultures were serially diluted (1:5), spotted on YPD plates and YPD plates with increasing concentrations of MMS using a 48-prong replicator and then incubated at 30 °C for 2 days. The exo1-FF447AA mutation shows no defect in the PRR pathway as this mutant is as resistant to MMS as wildtype and does not show any genetic interaction with the redundant (A) REV3- or (B) RAD9-dependent branches of PRR.

Figure 4

The exo1-FF447AA mutant is functional for PRR. Overnight saturated cultures were serially diluted (1:5), spotted on YPD plates and YPD plates with increasing concentrations of MMS using a 48-prong replicator and then incubated at 30 °C for 2 days. The exo1-FF447AA mutation shows no defect in the PRR pathway as this mutant is as resistant to MMS as wildtype and does not show any genetic interaction with the redundant (A) REV3- or (B) RAD9-dependent branches of PRR.