RESUMO
Loss of RAD52 is synthetically lethal in BRCA-deficient cells, owing to its role in backup homologous recombination (HR) repair of DNA double-strand breaks (DSBs). In HR in mammalian cells, DSBs are processed to single-stranded DNA (ssDNA) overhangs, which are then bound by replication protein A (RPA). RPA is exchanged for RAD51 by mediator proteins: in mammals, BRCA2 is the primary mediator; however, RAD52 provides an alternative mediator pathway in BRCA-deficient cells. RAD51 stimulates strand exchange between homologous DNA duplexes, a critical step in HR. RPA phosphorylation and dephosphorylation are important for HR, but its effect on RAD52 mediator function is unknown. Here, we show that RPA phosphorylation is required for RAD52 to salvage HR in BRCA-deficient cells. In BRCA2-depleted human cells, in which the only available mediator pathway is RAD52 dependent, the expression of a phosphorylation-deficient RPA mutant reduced HR. Furthermore, RPA-phosphomutant cells showed reduced association of RAD52 with RAD51. Interestingly, there was no effect of RPA phosphorylation on RAD52 recruitment to repair foci. Finally, we show that RPA phosphorylation does not affect RAD52-dependent ssDNA annealing. Thus, although RAD52 can be recruited independently of RPA's phosphorylation status, RPA phosphorylation is required for RAD52's association with RAD51 and its subsequent promotion of RAD52-mediated HR.
Assuntos
Reparo do DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Estafilocócica A/metabolismo , Reparo do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Humanos , Fosforilação , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Reparo de DNA por Recombinação/genética , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Studies of seizure management in the pediatric inpatient setting are needed. Seizures recorded by video EEG provide an opportunity to quantitatively evaluate acute management. We observed variation in delivery of standardized seizure safety measures (seizure first aid) during epilepsy monitoring unit admissions at our hospital. Our goals were to increase consistency and speed of seizure first aid and neurologic assessment in acutely seizing patients. METHODS: Using a root cause analysis, we identified major factors contributing to variation in seizure management and key drivers for improvement. Targeted interventions, centered around a protocol for acute seizure management, were implemented through quality improvement methodology. The primary outcome was correct performance of standardized seizure first aid and neurologic assessment. Secondary outcomes were time intervals to each assessment. Run charts were used to analyze primary outcomes, and statistical control charts were used for secondary outcomes. Nursing confidence in seizure management was determined through pre- and postsurveys and analyzed with the χ2 test. RESULTS: Thirteen seizures were evaluated in the preintervention phase and 10 in the postintervention phase. Completed components of seizure first aid increased from a median of 3 of 4 to 4 of 4; completed components of neurologic assessment increased from a median of 2 of 4 to 4 of 4. Responses to acute seizures were faster, and nursing confidence increased. CONCLUSIONS: A collaborative quality improvement effort between physicians and nurses led to prompt and correct delivery of seizure first aid by first responders. These relatively simple interventions could be adapted broadly to improve acute seizure management in the pediatric inpatient setting.
