RESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1005792.].
RESUMEN
Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.
Asunto(s)
Reparación del ADN/genética , Replicación del ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Cromátides/genética , Cromátides/efectos de la radiación , Inestabilidad Cromosómica/efectos de la radiación , Cromosomas/genética , Cromosomas/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Anemia de Fanconi/patología , Inestabilidad Genómica/genética , Inestabilidad Genómica/efectos de la radiación , Humanos , ARN Interferente Pequeño , Rayos UltravioletaRESUMEN
After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Primasa/fisiología , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , ADN/efectos de la radiación , Enzimas Multifuncionales/fisiología , Recombinasa Rad51/fisiología , Rayos Ultravioleta , Ciclo Celular , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Progresión de la Enfermedad , Relación Dosis-Respuesta en la Radiación , Células HeLa , Humanos , Proteína Homóloga de MRE11 , ARN Interferente Pequeño/metabolismoRESUMEN
Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.