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(Toronto – June 28, 2012) Since our cells’ responses to DNA damage act as a stop-gate against diseases such as cancer, researchers worldwide are increasingly honing in on the specific mechanisms by which the “emergency response” to DNA damage unfolds.
To further this understanding, Mount Sinai Hospital researchers in the lab of Lunenfeld Senior Scientist Dr. Dan Durocher have uncovered how the ubiquitin-dependent hierarchy of protein recruitment is established at DNA damage sites—a discovery that deepens understanding of genetic “protection” responses, and which opens the door to new, more sophisticated cancer therapies.
The study was published online today in Molecular Cell, and follows earlier work by Dr. Durocher in 2009, in which they identified key players in the DNA damage response mediated by ubiquitin signaling.
The response to DNA double-stranded breaks (DSBs) entails the hierarchical recruitment of proteins to DNA damage sites and is orchestrated by ATM-dependent phosphorylation and RNF8/RNF168-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs).
“In this study, we sought to understand how the RNF8 pathway employs ubiquitin and its recognition by UBDs to build the hierarchical recruitment of DNA repair factors to DNA double-strand breaks,” said Stephanie Panier, a graduate student in the lab of Dr. Durocher, and first author of the study. “But until now, we did not understand the exact means by which UBD-bearing proteins select their cognate ubiquitylated ligands in this pathway.”
Panier explained that they focused their work on RNF168, whose recruitment to DNA damage sites is dependent on its UBDs, and on RNF169—a paralog of RNF168. The observation that RNF169, despite being homologous to RNF168, lies downstream of the latter in the DSB response provided a case study for investigating how the ubiquitin-dependent hierarchy of protein recruitment is established in this signaling pathway.
The investigators used a combination approach of cell biology and biochemistry that involved microscopy-based deletion mapping and in vitro pull-down and ubiquitin binding assays. They showed that RNF168 and RNF169 accumulate sequentially at sites of DNA damage through the use of bipartite functional modules that contain one or more UBDs and an adjacent peptide motif called LRM, which provides target specificity. 
The researchers also mapped LRM motifs found in other proteins that rely on ubiquitin recognition for their recruitment to sites of DNA damage. One of these proteins is RAP80, which helps recruit the DNA repair factor BRCA1 to the DSBs. (BRCA1 is frequently mutated in breast cancer.)
Interestingly, results also showed the possibility to ‘re-program’ UBDs that are unrelated to the to the DNA damage response to accumulate at sites of RNF168 ubiquitylation, by simply fusing the UBD to the LRM motif of RNF169.
“Our results provide new insight into how the recognition of distinct ubiquitylated ligands helps to establish the spatiotemporal order of signaling events in the DNA damage response. We know that UBDs have evolved to recognize ubiquitin, but they don’t contain much intrinsic specificity for the actual ubiquitylated ligand. The addition of an adjacent targeting motif basically provides the sequence context that is required for ubiquitin-dependent protein interactions,” said Panier. “I suspect that this may be how other ubiquitin-dependent signaling pathways are organized as well, although additional work is required to show that this is indeed the case.”
Panier added that her team’s next steps will be to use LRM motifs to identify additional ubiquitylated proteins that are important in the DNA DSB response pathway.
The study was supported by the Canadian Institutes of Health Research.
Earlier work by Dr. Durocher identified RNF8 and RNF168, two key players in the DNA damage response mediated by ubiquitin signaling. RNF168 has also been shown to be mutated in RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder. Dr. Durocher and his colleagues found how the deubiquitylase enzyme OTUB1 inhibits UBC13, a protein that acts with RNF168 to amplify the RNF8-dependent ubiquitination. That discovery also improved understanding of familial breast and ovarian cancer, as the research showed that OTUB1 inhibits the action of BRCA1, a DNA repair protein often mutated in these cancers.

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