Histone

Gene

Cell biology

Biology

Ubiquitin ligase

Cancer research

Biochemistry

Ubiquitin

Genetics

Chromatin

Ubiquitin-conjugating enzyme

DNA repair

Enzyme

Ubiquitin-Protein Ligases

Chemistry

DNA damage

Deubiquitinating enzyme

Journal of Biological Chemistry

Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.Background: The RNF8/RNF168 E3 ligase cascade promotes ubiquitin-dependent protein assembly at DNA double-strand breaks (DSBs).Results: An overexpression screen identified new deubiquitylating enzymes (DUBs) opposing the RNF8/RNF168 pathway in human cells.Conclusion: USP44 counteracts RNF168-dependent ubiquitylation of proteins at DSB sites, including histone H2A.Significance: Identification of antagonizers of the RNF8/RNF168 pathway is crucial for understanding how cells regulate DSB repair. Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses. Background: The RNF8/RNF168 E3 ligase cascade promotes ubiquitin-dependent protein assembly at DNA double-strand breaks (DSBs). Results: An overexpression screen identified new deubiquitylating enzymes (DUBs) opposing the RNF8/RNF168 pathway in human cells. Conclusion: USP44 counteracts RNF168-dependent ubiquitylation of proteins at DSB sites, including histone H2A. Significance: Identification of antagonizers of the RNF8/RNF168 pathway is crucial for understanding how cells regulate DSB repair.

The Deubiquitylating Enzyme USP44 Counteracts the DNA Double-strand Break Response Mediated by the RNF8 and RNF168 Ubiquitin Ligases