Dataset Information

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

ABSTRACT: During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

SUBMITTER: Su GC

PROVIDER: S-EPMC5291256 | biostudies-other | 2016 Jul

REPOSITORIES: biostudies-other

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Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

Su Guan-Chin GC Yeh Hsin-Yi HY Lin Sheng-Wei SW Chung Chan-I CI Huang Yu-Shan YS Liu Yi-Chung YC Lyu Ping-Chiang PC Chi Peter P

Nucleic acids research 20160430 13

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of AD ...[more]

PMID: 27131790

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Project description:BackgroundRAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.MethodsTo investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach.ResultsHere, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion.ConclusionsOur study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Dataset Information

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

Publications

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

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