Lack of superoxide dismutase in a rad51 mutant exacerbates genomic instability and oxidative stress-mediated cytotoxicity in Saccharomyces cerevisiae

Highlights

• SOD1 inhibition can be used for selective killing of cancer cells deficient in homologous recombination pathway.

• Deletion of RAD51 and SOD1 does not show synthetic lethality in budding yeast.

• Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51.

• Accumulation of Rad51 deficiency-mediated DSB lesions increases intracellular ROS levels in the absence of Sod1.

• DSB repair pathways and ROS response signaling have significant mutual genetic crosstalk.

Abstract

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.

Introduction

Aerobic organisms are unavoidably exposed to intrinsic and extrinsic stress that threatens genomic integrity and cell viability throughout their lifespan. These insults include direct DNA strand breaks and the frequent occurrence of cytotoxic damage mediated by reactive oxygen species (ROS). Although physical DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs), is mostly responsible for cell lethality, oxidative DNA lesions created by endogenous ROS and oxidizing chemicals are also implicated in a large proportion of structural mutations and genomic instability that might eventually cause cancer, aging, and a variety of degenerative disorders [1], [2], [3]. To deal with these adverse conditions, cells have evolved highly coordinated processes such as various oxidative stress signaling systems and DNA damage response (DDR) pathways.

Mounting evidence suggests a highly involved mutual influence between ROS signaling and DDR pathways. ROS-induced DNA adducts, such as modified bases and abasic DNA sites, occur as often as ~10,000 times per cell every day [4]. Oxidative DNA damage, including the occasional production of SSBs, is highly mutagenic but lesions are not considered fatal because they would be efficiently processed with comparative ease by base excision repair (BER) and nucleotide excision repair (NER) [5], [6]. Cells deficient in the BER and NER pathway show not only elevated genomic instability with a higher mutation frequency but also greatly induced intracellular ROS levels, indicating that accumulated DNA damage is inversely capable of causing oxidative stress in the cell [7], [8]. Unrepaired alkylating DNA damage induces the nuclear accumulation of Yap1, a ROS-responsive transcription factor, which reveals a mechanistic link between BER-mediated DNA damage repair and oxidative stress signaling [9].

DNA DSBs arise approximately 10–50 per cell each day [10]. Although much less frequent than oxidative DNA damage or SSBs, DSBs are highly toxic to genome fidelity eventually leading to cell death because they can cause detrimental deletions, translocations, and critical fusions in the chromosome. DSBs in the G1 phase can be repaired by non-homologous end joining (NHEJ) mainly due to the compact chromatin and lack of sister chromatids. During relatively long S and G2 phases, however, replication-associated homologous recombination (HR) becomes predominant and crucial for DSB repair in yeast and especially in mammalian cancer cells, which are typified with uncontrolled cell division [11], [12]. Rad51, the homolog of E. coli RecA recombinase and a member of Rad52 epistasis group, is a central player in the HR pathway, searching for homology and strand pairing for recombination. Replication defects and chromosomal instability are induced in rad51 mutants [13], [14].

A genome-wide analysis of synthetic lethal interactions in budding yeast has disclosed novel genetic interactions among functionally independent genes of key proteins in the HR pathway and responders to oxidative stress such as Sod1 and Tsa1 [15]. Synthetic lethality refers to a type of genetic interaction that occurs when a combination of loss-of-function perturbations in two or more genes results in cell death, whereas a mutation in only one of these genes does not affect viability at all [16]. The synthetic lethal paradigm between the Rad52 epistasis group members and Sod1 has been proposed as a promising anticancer therapeutic since selective apoptosis was induced in RAD54B-deficient human colorectal cancer cells by Sod1 inhibition [17], [18]. In addition to its conventional action as a superoxide scavenger, Sod1 plays an important role as a transcription factor in the nucleus through direct phosphorylation by Dun1, an effector kinase in damage checkpoint signaling [19]. Moreover, cells with no functional ataxia-telangiectasia mutated (ATM) protein kinase are hypersensitive to oxidative stress, and the damage checkpoint activation mediated by Mec1, a yeast ATR homolog, is downregulated by the lack of Sod1, suggesting the existence of genetic and biochemical connections between cellular redox regulators and DDR pathways [20], [21], [22]. In a previous report, we revealed that Yap1 and Skn7, two transcription factors in charge of the oxidative stress response with various antioxidant enzymes, are highly implicated in the Rad51-mediated HR pathway in terms of a strategic defense mechanism against oxidative stress and DNA damaging insults [23].

In this study, unlike in previous reports derived from a high-throughput dataset, we show that the additional deletion of SOD1, a downstream target of Yap1 and Skn7, in an HR-defective rad51 mutant background is not synthetic lethal, but adversely affects the growth and genomic integrity of yeast cells with increased mutation frequency and activated damage checkpoint signaling. Importantly, we find that this novel role of Sod1 associated with Rad51 might be dependent on its conventional catalytic activity as an antioxidant and on the capability of intracellular translocation between cytosol and the nucleus. Accumulation of DSB damage in the rad51 sod1 double mutant inversely induces elevation of intracellular ROS levels. Altogether, our results suggest a specific genetic association between antioxidant defense signaling and the DNA DSB repair pathway, which provides a cooperative mechanism to maintain genome integrity and cell survival.