RESUMEN
Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fragmentos de Péptidos/metabolismo , Mapeo de Interacción de Proteínas , Subunidades de Proteína/metabolismo , Proteína de Replicación A/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Daño del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosforilación , Proteína de Replicación A/químicaRESUMEN
Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.
Asunto(s)
Cisplatino/metabolismo , Aductos de ADN/química , ADN/metabolismo , Proteína de Replicación A/metabolismo , Cisplatino/química , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , ADN/síntesis química , ADN/química , Aductos de ADN/metabolismo , Polarización de Fluorescencia , Humanos , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/metabolismo , Proteína de Replicación A/químicaRESUMEN
Cardiac trauma fatalities occur in 22% of victims of motor vehicle crash. Blunt cardiac trauma may result in myocardial rupture. Mechanisms of injury include compression, deceleration, laceration, and "hydraulic ram" effect. Diagnosis is difficult because of coexisting injuries and the lack of evidence-based protocols. Physiological changes in the elderly and the presence of comorbid illness result in poor survival rates when myocardial rupture occurs. Trauma nurses must be prepared to provide end-of-life care related to the poor outcome associated with myocardial rupture. This case study reports a right ventricular rupture in an elderly patient involved in a frontal motor vehicle crash.