UHRF1 is a sensor for DNA interstrand crosslinks and recruits FANCD2 to initiate the Fanconi anemia pathway.
Bottom Line: Knockdown cells display a drastic reduction in FANCD2 foci formation.Based on these results, we describe a mechanism of ICL sensing and propose that UHRF1 is a critical factor that binds to ICLs.In turn, this binding is necessary for the subsequent recruitment of FANCD2, which allows the DNA repair process to initiate.
Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.Show MeSH
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Mentions: Given that UHRF1 interacts directly with ICLs in vitro and is required for proper foci formation of FANCD2 in vivo, we speculated that UHRF1 itself is recruited to crosslinked DNA in vivo, and that this triggers the chromatin recruitment of FANCD2. To test this hypothesis directly, we turned to live-cell imaging using fluorophore-tagged proteins. UHRF1 and FANCD2 were stably expressed in HeLa cells as fusion proteins with mCherry and EGFP, respectively. We introduced ICLs with a localized laser stripe after incubating the cells with TMP (Thazhathveetil et al., 2007). We observed that UHRF1 was recruited to ICLs very quickly and formed a clear stripe within 30 s. FANCD2 was also recruited, albeit slightly more slowly than UHRF1, and formed a visible stripe within 5 min (Figure 5A). Importantly, there was no recruitment of either one of the proteins in the absence of TMP (Figure 5B). These data encouraged us to directly test, in live cells, whether UHRF1 mediates the recruitment of FANCD2 to ICLs. Using control and UHRF1 knockdown cells, we assessed the recruitment of FANCD2 to ICLs in the presence and absence of UHRF1. Strikingly, we found that knockdown of UHRF1 completely abolished FANCD2 recruitment (Figure 5C).
Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.