Abstract
Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumorigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2 enzymes for impacts on cellular DSB responses. With a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3 ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Carrier Proteins / genetics
-
Carrier Proteins / metabolism*
-
Cell Cycle / genetics
-
Cell Line, Tumor
-
Cell Survival / genetics
-
DNA Breaks, Double-Stranded / drug effects
-
DNA Breaks, Double-Stranded / radiation effects
-
Endodeoxyribonucleases
-
Green Fluorescent Proteins / genetics
-
Green Fluorescent Proteins / metabolism
-
HEK293 Cells
-
Humans
-
Immunoblotting
-
Microscopy, Confocal
-
Nuclear Proteins / genetics
-
Nuclear Proteins / metabolism*
-
RNA Interference
-
Recombinational DNA Repair*
-
Ubiquitin-Conjugating Enzymes / genetics
-
Ubiquitin-Conjugating Enzymes / metabolism*
-
Ubiquitin-Protein Ligases / genetics
-
Ubiquitin-Protein Ligases / metabolism*
-
Ubiquitination
Substances
-
Carrier Proteins
-
Nuclear Proteins
-
Green Fluorescent Proteins
-
UBE2D1 protein, human
-
UBE2D2 protein, human
-
UBE2D3 protein, human
-
Ubiquitin-Conjugating Enzymes
-
RNF138 protein, human
-
Ubiquitin-Protein Ligases
-
Endodeoxyribonucleases
-
RBBP8 protein, human