Figure 1.
Mgm101 interacts with Mph1 and MutSα.
(A) LC-MS. Prominent peaks from LC analysis with intensity above 400 were subjected for MALDI-TOF analysis. (B) Co-IPs. Reciprocal immunoprecipitations using FLAG-tagged Mgm101 expressed in cells with chromosomally 6xHis-tagged Msh2, Msh6 or Mph1. The empty vector transformants (wild type) were used as controls. A portion of the lysates (1%) was immunoblotted to show input proteins (left-hand panel). (C) The elution profiles of Mgm101-Myc, Mph1-FLAG and Msh6-HA as well as Msh2 following Superdex 200 chromatography reveal coincident elution in a high molecular weight complex at around 670 kDa, suggesting that they associate in a high molecular-weight complex, marked with an asterisk.
Figure 2.
Pso2 and Mph1-Mgm101-MutSα control redundant ICL repair pathways.
(A to G) Analysis of the ICL sensitivity of combinations of PSO2 with MPH1, MGM101, MSH6 and MSH2 gene disruptions, and an mph1K113Q mutant. The ICL-inducing agent was HN2. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean.
Figure 3.
Pso2 and Mph1-Mgm101-MutSα protect against ICL-induced GCRs.
(A) Left panel - schematic of the genomically-integrated (at the HXT13 locus on chromosome V) substrate used to measure GCRs. Simultaneous loss of both the CAN1 and URA3 genes can only occur via GCRs, producing cells able to form colonies able to grow on media containing canavanine and 5-FOA. See text for further details. Right panel - GCRs rates in wild type, pso2, mgm101, mph1 and their respective double mutants, both spontaneously occurring and induced by a sub-lethal dose of HN2 (10 mM). Rates were determined as described in the Materials and Methods section. (B) Accumulation of DSBs in HN2-treated wild type, pso2, pso2 msh2, mgm101, pso2 mgm101 and pso2 mgm101 msh2 cells. Exponentially growing cells were treated with 100 mM HN2 for 2 hours at 28°C, or mock-treated (U) with water, and subsequently allowed to repair in minimal medium for 2, 4 and 24 hours. The U24 sample was mock-treated allowed to repair for 24 hours. Samples were analysed on PFGE gels. (C) Rad53 phosphorylation following treatment with 100 mM HN2 and up to 2 hours recovery in wild type, pso2, msh2, mph1, pso2 msh2 and pso2 mph1 mutants.
Figure 4.
HRR and NHEJ mediated repair of ICL-associated DSBs is precluded in cells lacking Pso2 and components of the Mph1-Mgm101-MutSα repair pathways.
(A–F) HN2 sensitivity of combinations of pso2, msh2, rad52 and yku70 disruptants treated with HN2. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean. (G) HN2-induced chromatin recruitment of Rad51 and Yku70 in wild type, pso2, msh2 and mph1 single and double disruptants. Mcm2 is shown as a loading control for chromatin associated protein. Chromatin bound material is labelled chr, and that from whole cell extract labelled wce.
Figure 5.
Exo 1 is involved in the pathways controlled by Pso2 and Mph1-Mgm101-MutSα.
(A and B) HN2 sensitivity of combinations of pso2 and exo1 disruptants, and exo1-D173A mutants treated with HN2. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean. (C) HN2-induced chromatin recruitment of Exo1-FLAG in wt, pso2, mph1 and pso2 mph1 disruptants. Mcm2 is shown as a loading control for chromatin associated protein. Chromatin bound material is labelled chr, and that from whole cell extract labelled wce. (D) HN2-induced chromatin recruitment of Rad51 in wild type cells, pso2 and exo1 single and double disruptants. Mcm2 is shown as a loading control for chromatin associated protein. Chromatin bound material is labelled chr, and that from whole cell extract labelled wce.
Figure 6.
The FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are both involved in the ICL repair pathway controlled by Mph1-Mgm101-MutSα.
(A–D) HN2 sensitivity analysis of cells disrupted for PSO2 and MSH2 in combination with the FANCJ-related factor CHL1 and FANCP related factor SLX4. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean.
Figure 7.
A model for the two major ICL processing pathways in budding yeast, one controlled by Pso2 and the other by Mph1-Mgm101-MutSα in collaboration with Exo1.
See associated text for details.