Screening a genome-wide S. pombe deletion library identifies novel genes and pathways involved in genome stability maintenance

Abstract

The maintenance of genome stability is essential for an organism to avoid cell death and cancer. Based on screens for mutant sensitivity against DNA damaging agents a large number of DNA repair and DNA damage checkpoint genes have previously been identified in genetically amenable model organisms. These screens have however not been exhaustive and various genes have been, and remain to be, identified by other means. We therefore screened a genome-wide Schizosaccharomyces pombe deletion library for mutants sensitive against various DNA damaging agents. Screening the library on different concentrations of these genotoxins allowed us to assign a semi-quantitative score to each mutant expressing the degree of sensitivity. We isolated a total of 229 mutants which show sensitivity to one or more of the DNA damaging agents used. This set of mutants was significantly enriched for processes involved in DNA replication, DNA repair, DNA damage checkpoint, response to UV, mating type switching, telomere length maintenance and meiosis, and also for processes involved in the establishment and maintenance of chromatin architecture (notably members of the SAGA complex), transcription (members of the CCR4-Not complex) and microtubule related processes (members of the DASH complex). We also identified 23 sensitive mutants which had previously been classified as "sequence orphan" or as "conserved hypothetical". Among these, we identified genes showing extensive homology to CtIP, Stra13, Ybp1/Ybp2, Human Fragile X mental retardation interacting protein NUFIP1, and Aprataxin. The identification of these homologues will provide a basis for the further characterisation of the role of these conserved proteins in the genetically amenable model organism S. pombe.

Figure 1

Example illustrating the semi-quantitative scoring system used in the screens. Mutants where assigned a sensitivity score according to the lowest concentration of the DNA damaging agent at which they show clear growth inhibition (numbers in white).

Figure 2

Graph showing the over-represented GO-terms at a significance level smaller than 0.005. Colours of the different GO-terms represent statistical significance (see legend in top right corner). Related GO-terms are connected by a line. Grey boxes include lists of genes associated with any of the GO-terms within the box.

Figure 3

Spot test confirming the sensitivity of 23 mutants isolated in the screen which have previously been classified as “sequence orphan” or “conserved hypothetical”. Yellow boxes indicate sensitivities as detected in the large scale screen.

Figure 4

Alignments between a) S. pombe SPCC576.12c, S. cerevisiae Ydl160c-a and mouse Stra13. b) Alignments between S. pombe SPCC1442.02 and S. cerevisiae YBP2 and YBP1. c) alignments between S. pombe SPBC16c6.03c, Candida albicans SC5314 and the human fragile X mental retardation interacting protein NUFIP1 (labelled hsFMRP_IP1). d) Alignments between S. pombe SPCC18.09c, S. cerevisiae HNT3, Aspergillus nidulans AN2139.2 and human Aprataxin. All alignments show the complete amino acid sequences. Black boxes indicate identical amino acids, grey boxes indicate similar amino acids.