Chromosomal integrity after UV irradiation requires FANCD2-mediated repair of double strand breaks

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.Fil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vallerga, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Radl, Daniela Betiana. Autoridad Regulatoria Nuclear; ArgentinaFil: Paviolo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Di Giorgio, Marina. Autoridad Regulatoria Nuclear; ArgentinaFil: Soria, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

UV-triggered p21 degradation facilitates damaged-DNA replication and preserves genomic stability

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.Fil: Mansilla, Sabrina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina; Fundación Instituto Leloir; Argentina;Fil: Soria, Gastón. Fundación Instituto Leloir. Laboratorio de Ciclo Celular y Estabilidad Genómica; Argentina;Fil: Vallerga, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina; Fundación Instituto Leloir. Laboratorio de Ciclo Celular y Estabilidad Genómica; Argentina;Fil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina; Fundación Instituto Leloir. Laboratorio de Ciclo Celular y Estabilidad Genómica; Argentina;Fil: Martínez López, Wilner. Fundación Instituto Leloir. Laboratorio de Ciclo Celular y Estabilidad Genómica; Argentina; Ministerio de Educación y Cultura. Instituto de Investigaciones Biológicas Clemente Estable; Uruguay;Fil: Prives, Carol. Columbia University. Department of Biological Sciences; Estados Unidos de América;Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina

Rad51 prevents Mre11-dependent degradation and excessive primpol-mediated elongation of nascent DNA after UV irradiation

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.Fil: Vallerga, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Mansilla, Sabrina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Bertolin, Agustina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentin

FANCD2 is activated but it is not required for cell survival after UV irradiation.

<p>A) Western blot (W.B.) of FANCD2 (D2) in U2OS and PD20 cells expressing D2 (PD20+D2). B) Flow cytometry analysis of U2OS cells transfected with control or D2 siRNA after UV irradiation (5 J/m²) and MMC (40 ng/ml). Samples were collected 72 hours after DNA damage induction C) Clonogenic assay in U2OS cells transfected with control and D2 siRNA and treated with the indicated doses of UV irradiation and MMC. D) Surviva (Cell titer Glo) assay in PD20 and PD20+D2 cells treated with the indicated doses of UV irradiation and MMC. In all cases, the survival rate was calculated with respect to untreated samples within the same curve. For each panel, three independent experiments were analyzed obtaining similar results. For all figures in this manuscript: significance of the differences are: *p<0.1; **p<0.01; ***p<0.001; when the p value is not shown the difference is not statistically significant. Error bars represent SEM (standard error of the mean).</p

FANCD2 facilitates the recruitment of Rad51 to UV-damaged DNA and the activation of sister chromatid exchanges.

<p>A) Schematics and representative image of the recruitment of Rad51 to UV-irradiated sub-nuclear regions (visualized with γH2AX staining). B) Rad51 recruitment to damaged nuclear regions in U2OS cells transfected with control and D2 siRNAs, and C) in PD20 and PD20+D2 samples after UV irradiation (5 J/m²). D) Representative panel and SCE quantification in U2OS cells transfected with control and D2 siRNA (1.5 J/m²). E) 53BP1 and γH2AX focal organization in control and UV-treated cells (5 J/m²) transfected with FANCD2 or control siRNA. F) Focal organization of 53BP1 after UV irradiation (5 J/m²- solid color columns) and MMC treatment (40 ng/ml- striped columns) in U2OS cells transfected with control and D2 siRNA. The percentages of cells with 53BP1 foci in both UV- and MMC-treated samples are expressed as folds compared to untreated cells. Fold increases with respect to controls are shown below in black (UV) and grey (MMC). Significant differences for UV-treatment are shown (for MMC, ***p<0.001 at 24hrs). Figures are representative of 3 independent experiments.</p

Chromosome aberrations caused by UV irradiation of FANCD2-depleted cells are completely reverted by NHEJ inactivation.

<p>A) Pulse field gel electrophoresis showing the levels of DSB formation after 24 hours of UV irradiation in U2OS transfected with the indicated siRNA. Data quantification is shown underneath the PFGE image. B) MN accumulation in binucleated cells; C) gaps and breaks and D) complex chromatidic exchanges. Two independent experiments were analyzed obtaining similar results.</p

FANCD2 prevents gross chromosome rearrangements after UV irradiation.

<p>A) Representative binucleated cell with MN. B) MN accumulation in U2OS cells transfected with control and D2 siRNA after UV irradiation (5 J/m²). C) W.B. showing the levels of Ubi-D2 in PD20, PD20+D2 and GM00637 cells. D) MN accumulation in PD20, PD20+D2 and GM00637 cells after UV irradiation (5 J/m²). E) Gaps + breaks and F) complex chromatidic exchange accumulation in U2OS transfected with control and D2 siRNA after UV irradiation (1.5 J/m²). Three independent experiments were analyzed obtaining similar results.</p

FANCD2 depletion does not modulate TLS or checkpoint markers after UV irradiation.

<p>A) W.B. showing the extent of PCNA ubiquitination in control and D2 depleted samples after the indicated doses of UV irradiation in U2OS cells. Images belong to lanes within the same gel and correspond to the same exposure. Quantification of Ubi-PCNA levels 6 hours post-UV is shown on the right. B) Percentage of U2OS cells with more than 10 GFP-Pol η foci at the indicated times after UV radiation (5 J/m²). C) W.B. showing phospho-Chk1 (S3545), Chk1, p53 and p21 levels in U2OS transfected with control and FANCD2 siRNAs. Images belong to lanes within the same gel and correspond to the same exposure. Quantifications of p-Chk1, p53 and p21 normalized to KU70 for the 6-hours´ time point are shown on the right. Figure is representative of 3 independent experiments for each panel.</p