The Fanconi anemia core complex is required for efficient point mutagenesis and Rev1 foci assembly - PubMed (original) (raw)

The Fanconi anemia core complex is required for efficient point mutagenesis and Rev1 foci assembly

Kanchan D Mirchandani et al. DNA Repair (Amst). 2008.

Abstract

Fanconi anemia (FA) is a chromosome instability syndrome characterized by congenital abnormalities, cellular hypersensitivity to DNA crosslinking agents, and heightened cancer risk. Eight of the thirteen identified FA genes encode subunits of a nuclear FA core complex that monoubiquitinates FANCD2 and FANCI to maintain genomic stability in response to replication stress. The FA pathway has been implicated in the regulation of error-prone DNA damage tolerance via an undefined molecular mechanism. Here, we show that the FA core complex is required for efficient spontaneous and UVC-induced point mutagenesis, independently of FANCD2 and FANCI. Consistent with the observed hypomutability of cells deficient in the FA core complex, we also demonstrate that these cells are impaired in the assembly of the error-prone translesion DNA synthesis polymerase Rev1 into nuclear foci. Consistent with a role downstream of the FA core complex and like known FA proteins, Rev1 is required to prevent DNA crosslinker-induced chromosomal aberrations in human cells. Interestingly, proliferating cell nuclear antigen (PCNA) monoubiquitination, known to contribute to Rev1 recruitment, does not require FA core complex function. Our results suggest a role for the FA core complex in regulating Rev1-dependent DNA damage tolerance independently of FANCD2, FANCI, and PCNA monoubiquitination.

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Figures

Figure 1. FANCA- and FANCG-, but not FANCD-deficient cells, are hypomutable in the supF assay

(A) Schematic representation of the supF assay used to investigate the mutation frequency of various Fanconi Anemia cell lines. Undamaged or UVC-irradiated supF plasmid pSP189 is transfected into a cell line of interest, recovered 48 hrs post-transfection by alkaline lysis, Dpn1 digested, and electroporated into the MBM7070 indicator bacterial strain. The ratio of white to total number of colonies is the mutation frequency. (B) FANCA- (GM6914), FANCG- (PD326) and (C) FANCD2- (PD20) deficient patient-derived fibroblasts and their cDNA corrected counterparts were analyzed in the supF assay as described in (A). Error bars indicate the standard deviation of the mean between three independent experiments.

Figure 2. Mutation spectra analysis of UVC-irradiated supF plasmids from FA-deficient and corrected cells

UVC-irradiated mutant supF plasmids retrieved from (A) FANCA-deficient and corrected and (B) FANCG-deficient and corrected patient-derived fibroblasts were sequenced. Only mutations within the structural supF gene are shown.

Figure 3. The FA core complex is required for efficient Rev1 foci assembly

(A) FANCA- (GM6914) and FANCG- (PD326) deficient patient-derived fibroblasts and their cDNA corrected counterparts, and (B) siRNA-treated Hela cells were transiently transfected with murine eGFP-Rev1. 20 hours post-transfection, cells were treated with 10J/m2 of UVC radiation and 16 hours later, fixed with paraformaldehyde and analyzed via fluorescence microscopy. The ratio of cells with greater than five foci to cells with eGFP-Rev1 expression is indicated on the Y axis. Error-bars represent standard error between three independent transfections. The foci counts were conducted blind to reduce observer bias. (C) FANCG-deficient patient-derived cells (PD326) and their cDNA corrected counterparts were analyzed for eGFP-Rev1 distribution in terms of foci-per-cells as in (A). (D) FANCG-deficient and corrected cells were transiently transfected with murine eGFP-Rev1 with or without HA-Ub-encoding plasmid DNA, treated with 2mM hydroxyurea for 12 hrs, and analyzed by western blotting with an anti-GFP antibody.

Figure 4. The FA core complex and Rad18 function in parallel DNA damage response pathways

(A) FANCG-deficient patient-derived fibroblasts (PD326) and their cDNA corrected counterparts were treated with 20J/m2 of UVC and harvested at various time points, followed by western blotting with anti-PCNA, anti-FANCD2, and anti-vinculin antibodies. (B) Rad18-mutant versus wild-type murine fibroblasts were treated with 60J/m2 UVC and harvested at various time points, followed by western blotting with anti-PCNA and anti-mouse FANCD2 antibodies. (C) FANCG-deficient patient-derived fibroblasts (PD326) and their cDNA corrected counterparts were treated with control, Rad6-, or Rad18-specific siRNAs for 72 hours. Cell survival was assessed with a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). The ratio of cell survival in FANCG-deficient versus corrected cells is indicated. The error-bars indicate standard error between three independent experiments.

Figure 5. The FA core complex-mediated assembly of Rev1 nuclear foci requires the BRCT domain of Rev1

(A) Schematic of the Rev1 polypeptide domain structure. (B) Bar graphs quantifying cells with eGFP-Rev1 foci. FANCG-deficient patient-derived fibroblasts (PD326) and corrected cells were transiently transfected with wild-type or mutant murine eGFP-Rev1 cDNAs. 20hrs post transfection, cells were treated with 10J/m2 UVC radiation and 8 hrs later, fixed with paraformaldehyde and analyzed by fluorescence microscopy. The ratio of cells with greater than five eGFP-Rev1 foci to cells with eGFP-Rev1 expression is indicated on the Y axis. At least 300 cells were counted for each data point. Error-bars represent the standard error between three independent experiments.

Figure 6. Rev1 deficient cells display MMC-induced chromosomal aberrations

HEK293T cells were treated with control or Rev1 specific siRNAs for 72 hrs, exposed to 15 ng/ml MMC for 48 hrs and analyzed for chromosomal aberrations.

Figure 7. A model depicting the regulation of Rev1-dependent DNA damage tolerance by the FA core complex, independently of FANCD2 and FANCI

Schematic representation of FA core complex-mediated regulation of Rev1. The FA core complex plays a well known role in monoubiquitinating FANCD2 and FANCI, leading to HR repair. The complex is also required for efficient Rev1 foci assembly. The mechanism underlying FA core complex-controlled regulation of Rev1 localization is unknown, but it appears to be independent of PCNA and Rev1 monoubiquitination as well as the UBM domain of Rev1 but requires the BRCT domain of Rev1.

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The Fanconi anemia core complex is required for efficient point mutagenesis and Rev1 foci assembly - PubMed (original) (raw)