Dual functions of Mdt1 in genome maintenance and cell integrity pathways in Saccharomyces cerevisiae

Abstract

Recent evidence indicates considerable cross-talk between genome maintenance and cell integrity control pathways. The RNA recognition motif (RRM)-and SQ/TQ cluster domain (SCD)-containing protein Mdt1 is required for repair of 3 -blocked DNA double-strand breaks (DSBs) and efficient recombinational maintenance of telomeres in budding yeast. Here we show that deletion of MDT1 (PIN4 /YBL051C ) leads to severe synthetic sickness in the absence of the genes for the central cell integrity MAP kinases Bck1 and Slt2/Mpk1. Consistent with a cell integrity function, mdt1 cells are hypersensitive to the cell wall toxin calcofluor white and the Bck1-Slt2 pathway activator caffeine. An RRM-deficient mdt1-RRM0 allele shares the severe bleomycin hypersensitivity, inefficient recombinational telomere maintenance and slt2 synthetic sickness phenotypes, but not the cell wall toxin hypersensitivity with mdt1 . However, the mdt1-RRM(3A) allele, where only the RNAbinding site is mutated, behaves similarly to the wild-type, suggesting that the Mdt1 RRM functions as a protein-protein interaction rather than a nucleic acid-binding module. Surprisingly, in a strain background where double mutants are sick but still viable, bck1 mdt1 and slt2 mdt1 mutants differ in some of their phenotypes, consistent with the emerging concept of flexible signal entry and exit points in the Bck1-Mkk1/2-Slt2 pathway. Overall, the results indicate that Mdt1 has partially separable functions in both cell wall and genome integrity pathways.

The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

“…Although this idea may seem surprising, there is increasing evidence for a direct cross-talk between the DNA checkpoint pathway and cellular morphogenesis. Thus, various checkpoint proteins contribute to cell wall architecture and maintenance of cell polarity [ 11 ], Cdk1 regulates Rad53 to orchestrate cellular morphogenesis during cell cycle [ 49 ], Rad53 interacts with septins in the bud neck and directs filamentous differentiation in response to genotoxic stress [ 50 ], Rad53 phosphorylates Slt2 to control the Slt2-dependent expression of the cell wall FKS2 gene in response to caffeine [ 51 ], the Mdt1 protein has partially separable functions in both the cell wall and genome integrity pathways [ 52 ], and we have observed a synthetic lethality between slt2 and rad53 mutations that is suppressed by sorbitol, which suggests that lethality is caused by a morphogenetic defect. In fact, bud morphogenesis control has been described as an output of DNA replication checkpoint activation [ 11 ], which clearly demonstrates the link between genome maintenance and cell morphogenesis.…”

Yeast hEST1A/B (SMG5/6)–Like Proteins Contribute to Environment-Sensing Adaptive Gene Expression Responses

“…To test if the structural similarities extend to similar protein functions, we monitored esl1Δ and esl2Δ single-null and esl1Δ esl2Δ double-null mutants for telomere-related and NMD-related defects (Pike and Heierhorst 2007; Traven et al 2010). Telomere length in several independent esl1Δ , esl2Δ and esl1Δ esl2Δ clones were within the range of the wild-type, in contrast to rad50Δ mutants, which were included as a control for very short but stable telomeres (Figure 2, A and B), indicating that ESL1 and ESL2 do not contribute to normal telomerase-dependent telomere length control.…”

“…Membranes were incubated with radioactively labeled probes, exposed to phosphorimager screens, and analyzed using Molecular Dynamics ImageQuant software. For analysis of telomere lengths, genomic DNA was subjected to Xho I restriction endonuclease digestion at 37° for 4 hr as described (Pike and Heierhorst 2007; Traven et al 2010). …”

“…However, it recently has been shown that the yeast NMD factor Ebs1 is the structural and functional ortholog of hEST1C (SMG7) (Luke et al 2007), and no yeast counterparts for hEST1A/B (SMG5/6) previously have been identified. During database mining attempts to identify potential cofactors of a yeast protein with DNA damage and telomere-related functions, Mdt1/Pin4 (Pike and Heierhorst 2007; Pike et al 2004; Traven et al 2010), we noticed that a large-scale two-hybrid screen (Uetz et al 2000) had found it to interact with two uncharacterized Est-one-homology and PIN domain–containing open reading frames Yil151c and Ykr096w. Here, we show that Yil151c and Ykr096w are structural orthologs of hEST1A-B/SMG5-6 and have thus named them Esl1 and Esl2 (ESL = EST/SMG–like).…”

TheSaccharomyces cerevisiaePtc1 protein phosphatase attenuates G2‐M cell cycle blockage caused by activation of the cell wall integrity pathway

“…1B). Thus, lack of PIN4 (also known as MDT1), which was initially related to DNA damage responses, causes hypersensitivity to CFW and caffeine, and this mutation presents severe synthetic sickness with mutations in the BCK1 and SLT2 genes (Traven et al, 2010). The product of the essential VRG4 gene, a GDP-mannose transporter into the lumen of Golgi apparatus, is also required for maintenance of CWI (Ellerton et al, 2008).…”