Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae

Highlights

• Binding-partners to Pso2 nuclease of S. cerevisiae were isolated by two-hybrid system.

• Sak1p interacts with the β-CASP domain of Pso2p and phosphorylates Pso2p in vitro.

• PSO2 and SAK1 showed epistatic interaction after exposure to ICL-inducing agents.

• PSO2 interacts epistatically with RAD52, RAD50 and XRS2 for ICL repair in LOG cells.

• PSO2 showed non-epistatic interaction with MRE11 and no interaction with YKU70.

Abstract

By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.

Introduction

DNA interstrand cross-links (ICLs) are extremely genotoxic lesions since they covalently link the two complementary DNA strands, thereby resulting in stalled transcription and DNA replication, and ultimately prevent segregation of chromosomes. ICLs can arise from reactions with endogenous chemicals, such as the lipid peroxidation product malondialdehyde, or from exposure to various clinical anti-tumor drugs (e.g., cisplatin, nitrosoureas, nitrogen mustards) and photo-therapy treatments (psoralen plus UVA light – PUVA therapy) [1], [2]. The presence of one non-repaired ICL can be lethal in bacterial and yeast cells [1] and in mammalian cells 40 non-repaired ICLs induce cell death [3].

Yeast mutants cells hypersensitive to ICL-inducing agents were identified in the early 1980s by two independent genetic screenings in Saccharomyces cerevisiae, which showed sensitivity to photoactivated psoralen (PSO2) [4] and sensitivity to nitrogen mustard (SNM1) [5]. It was later demonstrated that these genes are allelic [6] and the nomenclature PSO2 was adopted to unify them [1]. pso2 yeast mutants have a unique feature, which is their hypersensitivity to photoactivated psoralen, bi- and polyfunctional alkylating compounds, while exhibiting only a wild-type (WT) level of sensitivity to monofunctional alkylating agents, ionizing radiation (IR), UVC light (254 nm) [7], [8] and HO endonuclease [9].

The yeast gene PSO2 encodes a 76 kDa nuclear protein that is believed to belong to a small group of proteins acting predominantly during ICL repair (for a review, see Brendel et al. [10]). Pso2 yeast protein has DNA 5′-exonuclease activity [11] and an endonuclease activity specific for DNA hairpin opening [12]. This nuclease activity is conferred by the metallo-β-lactamase hydrolase together with the β-CASP (metallo-β-lactamase-associated CPSF Artemis SNM1/PSO2) domain [13], [14]. The β-CASP domain consists of a conserved region in the C-terminal moiety that is unique to the PSO2 gene family and is predicted to be a nucleic acid binding domain [11], [13], [15], [16], [17]. The expression of yeast PSO2 mRNA is constitutively low, with approximately 0.3 transcripts/cell [18], but is induced about four times by ICL-inducing agents in exponentially growing cells [19]. Over-expression of PSO2 does not lead to an increased resistance to nitrogen mustard and cisplatin [20].

Five human orthologs for PSO2 were identified: SNM1A, SNM1B/Apollo, and SNM1C/Artemis – which have roles in DNA metabolism and cell cycle regulation (reviewed by [14], [21], [22]; CPSF73 and ELAC2 – involved in RNA processing [15], [21]. SNM1Ap is the functional mammal homolog of yeast Pso2p [23], [24] and has roles in mediating resistance to some cross-linking drugs and in the maintenance of genome stability following ICL formation. SNM1B/Apollo protein possesses a 5′-exonuclease activity and plays a role in telomere maintenance [16]. SNM1C/Artemis alone also has 5′-exonuclease activity, while the Artemis:DNA-PK complex endonucleolytically cleaves 5′ and 3′ overhangs and has hairpin-opening activity [25], acting in V(D)J recombination and immune competence; its deficiency results in a severe combined immunodeficiency, associated with increased cellular radio-sensitivity (RS-SCID) phenotype [26].

An intriguing aspect of ICL repair is that several DNA repair pathways have to work together in order to remove or bypass this lesion. In general terms, the pathways involved in ICL repair in S. cerevisiae are defined by homologous recombination (HR), translesion bypass synthesis (TLS) and nucleotide excision repair (NER) [8], [9], [27], [28], [29]. The involvement of Pso2 protein with these pathways still presents incompletely understood features. In yeast as in mammalian cells, cellular response to ICLs is cell cycle dependent [30]. The majority of ICLs are thought to be repaired during S phase, when the collapse of a replication fork encountering an ICL triggers the repair machinery [30], [31]. Indeed, an ICL-stalled replication fork results in the formation of a double strand break (DSB) that represents a severe damage to the cell [32] and must be properly repaired. In G1 phase, the ICL can be recognized by a stalled RNA polymerase during transcription or by NER factors [33], and an ICL processing pathway that includes NER and TLS was shown to predominate repair during this phase of the yeast cell cycle [29].

It is well-established that enzymes of NER start processing ICL-containing DNA, but the repair process is post-incisionally blocked and DSBs are accumulated in absence of functional Pso2p, preventing the reconstitution of high molecular weight DNA in yeasts [34], [35]. This points to a Pso2p function in ICL repair downstream of the incision event [9]. Moreover, the specific requirement for Pso2p in ICL repair suggests that DSBs formed during this process may be different from other forms of DNA breaks. Our bioinformatic analyses suggested Pso2p also to be a specific endonuclease related to the opening of hairpin structures that arise during DNA replication in the presence of ICLs [10] and, indeed, Pso2p was recently found to be a structure-specific DNA hairpin opening endonuclease, whose activity was required for repair of chromosomal breaks containing closed hairpin ends [12]. Thus, besides its 5′-exonuclease function in DNA end resection [11], Pso2p may function in the processing of DNA ends to generate proper substrates for the repair process, as has already been proposed by other authors [34], [36]. However, its precise function has remained elusive and there are still many aspects to be elucidated about Pso2p function in ICL repair.

The role of Pso2p in yeast ICL-repair could be better understood if its interaction with other proteins were known. Therefore, the aim of this study was to identify potential Pso2p binding partners using the yeast two-hybrid system (THS). Here we present a collection of potential binding partners of Pso2p and demonstrate in vitro that Pso2p is a phosphorylation target for protein kinase Sak1 (encoded by the open reading frame YER129w), a temperature-sensitive suppressor of DNA polymerase alpha (Pol α) mutations [37] and also an activating kinase of the Snf1 protein under cellular stress conditions [38]. Furthermore, we show that Pso2p interacts epistatically with Sak1p after exposure to DNA-ICL inducing agents and that both proteins immunoprecipitate after 8-MOP photoaddition. Besides, survival assays in exponential cell growth revealed an epistatic interaction of PSO2 with XRS2, RAD50 and RAD52, while a non-epistatic interaction was observed with MRE11 gene and no interaction was detected with YKU70 and DNL4 genes in DNA-ICL containing yeast cells.