Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway - PubMed (original) (raw)

Kimiyo N Yamamoto et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitin-binding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4(-/-) cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNA-damage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

SLX4 is essential for cellular proliferation. (A) RT-PCR analysis of SLX4 transcripts in SLX4−/−tetSLX4 cells using primers hybridizing with exons 14 and 18. β-actin served as a loading control. (B) Growth curves of the indicated cells. The tetSLX4 transcription was active without doxycyclin (ON) and was inhibited after the addition of doxycyclin (OFF). (C) Representative cell cycle profiles of indicated cells. Cells were pulse-treated with BrdU for 10 min, then subjected to flow cytometric analysis. Propidium iodide staining is displayed on the _x_-axis (linear scale), and BrdU incorporation is displayed on the _y_-axis (logarithmic scale). The square on the left side indicates apoptotic cells (sub-G1 fraction), the rectangle at the bottom left represents G1 phase cells, the arch represents S phase cells, and the square at the bottom right represents G2/M phase cells. Numbers indicate the relative percentage of cells in each gate, including dead cells in the sub-G1 fraction. The experiment was repeated three times; values are mean ± SD. (D) Number of chromosomal aberrations per 50 metaphase nuclei of indicated cells.

Fig. 2.

Fig. 2.

The UBZ domain of SLX4 is required for cellular tolerance to ICL-inducing agents. Cells were exposed to the indicated genotoxic agents for 72 h, a period during which WT cells were able to divide nine times in the absence of exogenous DNA damage. The _x_-axis represents the concentration of the genotoxic agents, and the _y_-axis represents the relative number of surviving cells at 72 h (logarithmic scale). Error bars represent SD from independent experiments.

Fig. 3.

Fig. 3.

Recruitment of SLX4 to DNA-damage sites thorough interaction between SLX4-UBZ domain and ubiquitylated FANCD2. (A) Localization of FANCD2, SLX4-GFP, and SLX4-UBZΔ-GFP in cells treated with MMC. Indicated cells were exposed to 500 ng/mL of MMC for 6 h. Fixed cells were stained with anti-FANCD2 antibody and visualized by fluorescence microscopy. The bar graph shows the percentage of colocalization of SLX4-GFP and FANCD2 foci. Cells displaying more than four colocalized foci were defined as foci-positive. (B) DT40 cells expressing the SLX4-GFP or SLX4-UBZΔ-GFP transgene were treated with 500 ng/mL of MMC for 6 h. Extracts of formaldehyde-fixed cells were incubated with an anti-GFP antibody, and the immunoprecipitates were analyzed with antibodies against FANCD2. SLX4-GFP interacts preferentially with the monoubiquitylated form of FANCD2.

Fig. 4.

Fig. 4.

Genetic relationship between FANCC− and SLX4-UBZΔ. (A) Sensitivity of cells with the indicated genotype to cisplatin and MMC. The drug concentrations are displayed on the _x_-axis (linear scale), and the relative number of surviving cells at 72 h is displayed on the _y_-axis (logarithmic scale). Error bars represent SD from three independent experiments. (B) Number of chromosomal aberrations per 50 metaphase nuclei from the indicated cells. Cells were treated with MMC (40 ng/mL for 24 h). _FANCC_−/SLX4-UBZΔ cells displayed a larger number of chromosomal aberrations than SLX4-UBZΔ or _FANCC_−/SLX4−/−tetSLX4 cells. P < 0.0001 for each.

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