The conserved Fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast

Abstract

DNA interstrand cross-links (ICLs) represent a physical barrier to the progression of cellular machinery involved in DNA metabolism. Thus, this type of adduct represents a serious threat to genomic stability and as such, several DNA repair pathways have evolved in both higher and lower eukaryotes to identify this type of damage and restore the integrity of the genetic material. Human cells possess a specialized ICL-repair system, the Fanconi anemia (FA) pathway. Conversely yeasts rely on the concerted action of several DNA repair systems. Recent work in higher eukaryotes identified and characterized a novel conserved FA component, FAN1 (Fanconi anemia-associated nuclease 1, or FANCD2/FANCI-associated nuclease 1). In this study, we characterize Fan1 in the yeast Schizosaccharomyces pombe. Using standard genetics, we demonstrate that Fan1 is a key component of a previously unidentified ICL-resolution pathway. Using high-throughput synthetic genetic arrays, we also demonstrate the existence of a third pathway of ICL repair, dependent on the SUMO E3 ligase Pli1. Finally, using sequence-threaded homology models, we predict and validate key residues essential for Fan1 activity in ICL repair.

Keywords: Cisplatin; Epistasis; Genetic screen; ICL; Schizosaccharomyces pombe; Synthetic array.

Fig. 1

Fan1 is a novel component of a pso2-independent interstrand crosslink repair pathway in S. pombe. (A) Sensitivity of combinations of fan1 and pso2 deletion mutants to cisplatin and MMC. Top panel: logarithmically grown cultures were spotted in four 1:10 serial dilutions starting from 10⁷ cells (first spot on the left) on YEA plates containing the agents in the amount indicated. Bottom panel: due to the short half-life of HN2, sensitivity to this drug was assessed by exposing 4 × 10⁷ cells from logarithmically growing cultures to the indicated dose. Error bars represent the standard error of the mean of three independent experiments. (B) Sensitivity of combinations of fan1, pso2 and rad13 deletion mutants to cisplatin (fan1-d: 14152N background). (C) Sensitivity of combinations of fan1, pso2 and rad51 deletion mutants. (D) Sensitivity of combinations of fml1 deletion mutants. rad3-d is used as a standard hypersensitive control for the efficacy of the agents used. UV treatment was included as a control for rad51 sensitivity. cispl, cisplatin; MMC, mitomycin C; HN2, bis(2-chloroethyl) methylamine; UV, ultra-violet irradiation.

Fig. 1

Fan1 is a novel component of a pso2-independent interstrand crosslink repair pathway in S. pombe. (A) Sensitivity of combinations of fan1 and pso2 deletion mutants to cisplatin and MMC. Top panel: logarithmically grown cultures were spotted in four 1:10 serial dilutions starting from 10⁷ cells (first spot on the left) on YEA plates containing the agents in the amount indicated. Bottom panel: due to the short half-life of HN2, sensitivity to this drug was assessed by exposing 4 × 10⁷ cells from logarithmically growing cultures to the indicated dose. Error bars represent the standard error of the mean of three independent experiments. (B) Sensitivity of combinations of fan1, pso2 and rad13 deletion mutants to cisplatin (fan1-d: 14152N background). (C) Sensitivity of combinations of fan1, pso2 and rad51 deletion mutants. (D) Sensitivity of combinations of fml1 deletion mutants. rad3-d is used as a standard hypersensitive control for the efficacy of the agents used. UV treatment was included as a control for rad51 sensitivity. cispl, cisplatin; MMC, mitomycin C; HN2, bis(2-chloroethyl) methylamine; UV, ultra-violet irradiation.

Fig. 2

The nuclease and the SAP domains of Fan1 are required for wild-type resistance to cisplatin. (A) Amino acid sequence alignment between HsFAN1 and SpFan1. Manually annotated ClustalW2 alignment (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The boxed regions indicate the conserved PD-(D/E)-XK nuclease motif . Asterisks indicate the residues mutated in our study (Leu159, Asp651, Glu666, Lys668 in the S. pombe homolog). These, plus additional mutants are listed in Fig. 3B (inset table). (B) Phyre2 sequence-threaded models of the spFAN1 SAP domain. Molecular ‘cartoon’ representations of the structural models based on PDB templates 2rnn and 2kvu. Key amino acids are additionally show in stick representation. The extent, quality and detail of each model is indicated by the inset amino acid sequence alignment and associated Phyre2 summary table. (C) Sensitivity of point mutations in the conserved residues of the nuclease domain to cisplatin and UV. A pso2-d background was used in order to compare the effect of the mutations to the hypersensitive double mutant fan1-d pso2-d. Logarithmically grown cultures were spotted in four 1:10 serial dilutions starting from 10⁷ cells (first spot on the left) on YEA plates containing the agents in the amount indicated. rad3-d is used as a standard hypersensitive control for the efficacy of the agents used. UV, ultra-violet irradiation; cispl, cisplatin; q. mutant, fan1-R160E R164E K171E R173E; N-term trunc, N-terminal truncation mutant d. Sensitivity of point and truncation mutants in the SAP domain to cisplatin. As described under (C).

Fig. 2

The nuclease and the SAP domains of Fan1 are required for wild-type resistance to cisplatin. (A) Amino acid sequence alignment between HsFAN1 and SpFan1. Manually annotated ClustalW2 alignment (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The boxed regions indicate the conserved PD-(D/E)-XK nuclease motif . Asterisks indicate the residues mutated in our study (Leu159, Asp651, Glu666, Lys668 in the S. pombe homolog). These, plus additional mutants are listed in Fig. 3B (inset table). (B) Phyre2 sequence-threaded models of the spFAN1 SAP domain. Molecular ‘cartoon’ representations of the structural models based on PDB templates 2rnn and 2kvu. Key amino acids are additionally show in stick representation. The extent, quality and detail of each model is indicated by the inset amino acid sequence alignment and associated Phyre2 summary table. (C) Sensitivity of point mutations in the conserved residues of the nuclease domain to cisplatin and UV. A pso2-d background was used in order to compare the effect of the mutations to the hypersensitive double mutant fan1-d pso2-d. Logarithmically grown cultures were spotted in four 1:10 serial dilutions starting from 10⁷ cells (first spot on the left) on YEA plates containing the agents in the amount indicated. rad3-d is used as a standard hypersensitive control for the efficacy of the agents used. UV, ultra-violet irradiation; cispl, cisplatin; q. mutant, fan1-R160E R164E K171E R173E; N-term trunc, N-terminal truncation mutant d. Sensitivity of point and truncation mutants in the SAP domain to cisplatin. As described under (C).

Fig. 3

The PEM-2 screen identifies a novel Pli1-dependent pathway of ICL repair. (A). Schematic representing the marker selection process throughout the PEM-2 high-throughput screen. The PEM-2 (Pombe Epistatic Mapper—2) approach is based on recessive resistance to the drug cycloheximide. Step1 (blue panel): construction of the fan1::natMX6 query mutant. Step 2 (green panel): screen of the Bioneer^® deletion mutant library. Mating and selection procedures ensure the maintenance of the three markers NAT^R, G418^R and cyh^R (at the native locus), conferring to the final double deletion mutant resistance to nourseothricin, geneticin and cycloheximide, respectively. See supplementary section and for further details. (B). Sensitivity to cisplatin of the combination of mutants hus1, rad1 and rad17 with fan1. Logarithmically grown cultures were spotted in four 1:10 serial dilutions starting from 10⁷ cells (first spot on the left) on YEA plates containing the agents in the amount indicated. rad3-d is used as a standard hypersensitive control for the efficacy of the agents used. The double mutants tested in this and the above experiments were derived from independently constructed single deletion mutants. fan1-d: 3909 and 14152 backgrounds. UV, ultra-violet irradiation; cispl, cisplatin. (C) Sensitivity to UV and cisplatin of the combination of pli1 and fan1 null mutants. As described under (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Pli1 acts on a pathway of ICL repair distinct from the fan1- and the pso2-dependent systems. (A) Sensitivity of pli1-deleted mutants combined with deletions of fan1 and pso2. fan1-d: 3909 background. Logarithmically grown cultures were spotted in four 1:10 serial dilutions starting from 10⁷ cells (first spot on the left) on YEA plates containing the agents in the amount indicated. rad3-d is used as a standard hypersensitive control for the efficacy of the agents used. Abbreviations used: UV, ultra-violet irradiation; cispl, cisplatin. (B) Proposed schematic of ICL resolution in S. pombe. The components of the various DNA repair pathways are shown in the relevant boxes, as assigned from the genetic analysis presented in this study. Left panel: possible roles for Fan1 in the Fan1-dependent resolution pathway. For simplicity, only the double fork model of ICL resolution is shown.