Activation of Checkpoint Kinase Chk1 by Reactive Oxygen Species Resulting from Disruption of wat1/pop3 in Schizosaccharomyces pombe

Abstract

DNA double-strand breaks are critical lesions that can lead to chromosomal aberrations and genomic instability. In response to DNA damage, Chk1, a serine/threonine kinase, is responsible for cell cycle arrest to prevent damaged cells from progressing through the cell cycle. Here, we report that the disruption of wat1, a WD repeat-containing protein, leads to the phosphorylation of Chk1. The double-deletion of chk1 and wat1 had a grave effect on the survival of fission yeast cells, and the spontaneous recombination rate was also high upon double-deletion of wat1 and chk1, as compared to the single-mutant. In the absence of wat1, the cells exhibited a high level of nuclear fragmentation that resulted in the accumulation of Rad22 yellow fluorescent protein foci. Furthermore, we show that wat1 is required for the regulation of the oxidative stress response. We observed elevated levels of reactive oxygen species (ROS) generation in wat1-null mutant that led to a high degree of propidium iodide staining at nonpermissive temperature. Based on the results presented here, we hypothesize that ROS production in wat1-null mutant cells generates DNA fragmentation that could trigger a checkpoint response and that, in the absence of checkpoint kinase Chk1, the cells exhibit severe growth defects leading to a synthetic lethal phenotype.

Critical role of Wat1/Pop3 in regulating the TORC1 signalling pathway in fission yeast S. pombe

“…Abrogation of Wat1 in tor2-287 mutant background leads to synthetic lethality Tor2, a component of TOR complex 1 (TORC1) is responsible for promoting vegetative growth and suppresses sexual development [4]. Earlier, we have shown the role of Wat1, an important component of TORC1 and TORC2 in oxidative stress response and sexual differentiation [20]. Further to analyse the genetic interaction between tor2 and wat1, we tried to construct a double mutant of wat1Δ with a tor2-287 mutant.…”

“…Fission yeast Wat1/Pop3, a WD repeats containing protein is an important component of both TORC1 and TORC2 and exhibits conditional synthetic lethality with chk1 knockout [19,20]. The mammalian LST8, also known as GβL (an ortholog of S. pombe Wat1), is also involved in nutrient and growth factormediated signalling to S6K1 [21].…”

“…It also affects the actin lament organization thus involved in the maintenance of cell shape [23] and is required for the maintenance of genomic stability and microtubule integrity [24]. More recently we have shown its active involvement during the oxidative stress response [20]. Budding yeast Lst8, an ortholog of Wat1 is required for the transportation of amino acid permeases on the cell surface [25].…”

Schizosaccharomyces pombe Assays to Study Mitotic Recombination Outcomes

“…The Whitby lab has subsequently been influential in designing several variations of the tandem repeat assay and have been able to explain some of the more complex outcomes. These assays have been used to identify and characterize genes involved in DNA damage repair [67][68][69][70][71][72][73][74][75][76][77][78]. The investigators initially designed a system similar to the Schuchert and Kholi assay except that instead of ura4 + they placed a his3 + marker between the ade6 alleles ( Figure 3C) [79].…”

Wat1/mLst8, a TOR complex protein regulates mitochondrial integrity and calcium ion homeostasis in fission yeastS. pombe

“…Its role in genomic stability and microtubule integrity has also been reported (Verma et al, 2014; Ochotorena et al, 2001). Previously, we have shown that the inactivation of Wat1 in fission yeast cells results in ROS accumulation that might affect the mitochondrial integrity (Ahamad et al, 2016). In this study, we investigated the role of Wat1 in the maintenance of mitochondrial homeostasis.…”