Genome-wide screens for sensitivity to ionizing radiation identify the fission yeast nonhomologous end joining factor Xrc4

Abstract

Nonhomologous end joining (NHEJ) is the main means for repairing DNA double-strand breaks (DSBs) in human cells. Molecular understanding of NHEJ has benefited from analyses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In human cells, the DNA ligation reaction of the classical NHEJ pathway is carried out by a protein complex composed of DNA ligase IV (LigIV) and XRCC4. In S. cerevisiae, this reaction is catalyzed by a homologous complex composed of Dnl4 and Lif1. Intriguingly, no homolog of XRCC4 has been found in S. pombe, raising the possibility that such a factor may not always be required for classical NHEJ. Here, through screening the ionizing radiation (IR) sensitivity phenotype of a genome-wide fission yeast deletion collection in both the vegetative growth state and the spore state, we identify Xrc4, a highly divergent homolog of human XRCC4. Like other fission yeast NHEJ factors, Xrc4 is critically important for IR resistance of spores, in which no homologous recombination templates are available. Using both extrachromosomal and chromosomal DSB repair assays, we show that Xrc4 is essential for classical NHEJ. Exogenously expressed Xrc4 colocalizes with the LigIV homolog Lig4 at the chromatin region of the nucleus in a mutually dependent manner. Furthermore, like their human counterparts, Xrc4 and Lig4 interact with each other and this interaction requires the inter-BRCT linker and the second BRCT domain of Lig4. Our discovery of Xrc4 suggests that an XRCC4 family protein is universally required for classical NHEJ in eukaryotes.

Keywords: Schizosaccharomyces pombe; XRCC4; nonhomologous end joining.

Figure 1

IR sensitivity screens identified xrc4 as a gene required for IR resistance of spores. (A) The procedure used to generate a mutant spore pool for the spore IR sensitivity screen. (B) Scatter plots of the log2(control/treatment) ratios from the two IR sensitivity screens. Genes are ordered on the x-axis according to their chromosomal positions. xrc4 and two known core NHEJ genes, pku80 and xlf1, are highlighted in red. (C) Survival curves of spores treated with different doses of IR.

Figure 2

Fission yeast Xrc4 is a homolog of human XRCC4 and budding yeast Lif1. (A) Multiple sequence alignment of the N-terminal conserved region of XRCC4 family proteins. The alignment was generated using the MAFFT-L-INS-i method (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013). Secondary structural elements of human XRCC4 (PDB 1ik9) and S. cerevisiae Lif1 (PDB 1z56) were visualized together with the sequence alignment using the ESPript 3.0 web server (http://espript.ibcp.fr/) (Gouet et al. 2003). (B) Phylogenetic tree based on the alignment in (A). The tree was constructed using the neighbor-joining (NJ) method (http://mafft.cbrc.jp/alignment/server/phylogeny.html) and visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The Arabidopsis homolog of XRCC4 was used as the outgroup to root the tree. Protein sequence accession numbers are gi|12081905 (Homo sapiens), gi|37589745 (Danio rerio), gi|9800643 (Arabidopsis thaliana), gi|7294937 (Drosophila melanogaster), gi|563290357 (Sclerotinia borealis), gi|75858908 (Aspergillus nidulans), gi|389638394 (Magnaporthe oryzae), gi|572283599 (Trichoderma reesei), gi|171690284 (Podospora anserina), gi|477536394 (Colletotrichum orbiculare), and gi|530775004 (Schizosaccharomyces japonicus), gi|295443012 (Schizosaccharomyces pombe), gi|528062605 (Schizosaccharomyces octosporus), gi|27948821 (Candida glabrata), gi|367016485 (Torulaspora delbrueckii), gi|254585561 (Zygosaccharomyces rouxii), gi|113913533 (Saccharomyces pastorianus), and gi|6321348 (Saccharomyces cerevisiae).

Figure 3

Xrc4 is required for classical NHEJ-mediated DSB repair. (A) Schematic of the ura4⁺ circularization assay. (B) Like lig4Δ and xlf1Δ, xrc4Δ causes a severe defect in circularizing the linear ura4⁺ DNA. The circularization efficiencies were normalized to that of the wild type. Error bars represent the SEM. (C) Schematic of the HO survivor assay. (D) Like pku70Δ and lig4Δ, xrc4Δ causes a reduction of HO survivor frequency. Error bars represent the SEM. (E) The HO repair junctions in xrc4Δ survivors share the same pattern as those in pku70Δ and lig4Δ survivors. The repair junction types are named as in Li et al. (2012). See Table S4 for all junctions with higher than 1% frequency in at least one of the four samples.

Figure 4

Xrc4 and Lig4 influence each other’s subcellular localization. (A) Lig4-GFP expressed from the P41nmt1 promoter can rescue the ura4⁺ circularization defect of lig4Δ. (B) Xrc4-mCherry expressed from the P41nmt1 promoter can rescue the ura4⁺ circularization defect of xrc4Δ. (C) The subcellular distribution of exogenously expressed Lig4-GFP and Xrc4-mCherry changed on co-expression. DNA was stained with Hoechst 33342.

Figure 5

Xrc4 and Lig4 physically interact with each other. (A) Xrc4-mCherry can be co-immunoprecipitated with Lig4-GFP. Coomassie staining of PVDF membrane after immunodetection was used to control for protein loading and blotting efficiency (Welinder and Ekblad 2011). (B) Xrc4 and Lig4 interact in the yeast two-hybrid assay and the interaction requires the inter-BRCT linker and the BRCT2 domain of Lig4.