Abstract
Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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BRCA2 Protein / genetics
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BRCA2 Protein / metabolism
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cell Line, Tumor
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DNA Damage*
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DNA Repair
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Gene Regulatory Networks
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Genome, Human / genetics*
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Heterogeneous-Nuclear Ribonucleoproteins / genetics*
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Heterogeneous-Nuclear Ribonucleoproteins / metabolism
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Histone Chaperones / genetics
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Histone Chaperones / metabolism
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Homologous Recombination*
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Humans
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Immunoblotting
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Microscopy, Fluorescence
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Models, Genetic
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Nuclear Pore Complex Proteins / genetics
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Nuclear Pore Complex Proteins / metabolism
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Poly(ADP-ribose) Polymerases / genetics
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Poly(ADP-ribose) Polymerases / metabolism
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RNA Interference
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RNA Precursors / genetics
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RNA Precursors / metabolism
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RNA, Small Interfering / genetics
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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Rad51 Recombinase / genetics
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Rad51 Recombinase / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Transcription Factors / genetics
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Transcription Factors / metabolism
Substances
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BRCA2 Protein
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Cell Cycle Proteins
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HIRA protein, human
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Heterogeneous-Nuclear Ribonucleoproteins
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Histone Chaperones
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NUP214 protein, human
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Nuclear Pore Complex Proteins
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Nuclear Proteins
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RBMX protein, human
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RNA Precursors
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RNA, Small Interfering
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RNA-Binding Proteins
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Transcription Factors
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UBN1 protein, human
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Green Fluorescent Proteins
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Poly(ADP-ribose) Polymerases
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Rad51 Recombinase