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. 2005 Jan 25;102(4):1110-5.
doi: 10.1073/pnas.0407796102. Epub 2005 Jan 13.

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair

Koji Nakanishi  1 , Yun-Gui Yang, Andrew J Pierce, Toshiyasu Taniguchi, Martin Digweed, Alan D D'Andrea, Zhao-Qi Wang, Maria Jasin
Affiliations

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair

Koji Nakanishi et al. Proc Natl Acad Sci U S A. .

Abstract

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca-/- cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.

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Figures

Fig. 1.
Fig. 1.
FA patient-derived cell lines have reduced HDR of a chromosomal DSB. (a) DSB reporter substrate. The recombination reporter DR-GFP is stably integrated into the genome of FA patient-derived cell lines. SceGFP is a GFP gene that contains an I-SceI endonuclease site within the coding region. Cleavage of the I-SceI site in vivo and repair by HDR directed by the downstream iGFP repeat results in GFP+ cells. (b and c) Flow cytometric analysis of FA-A cells (GM6914) and FA-G cells (EUFA326), demonstrating increased HDR after FANCA and FANCG complementation, respectively. FA cell lines were infected with the cognate FANC gene-expressing retrovirus or a nonexpressing virus (vector). Subsequently, they were transfected with an I-SceI expression vector (Center and Right) or an empty vector (Left). (d) HDR in FA-A and FA-G cells after I-SceI expression. The difference between the mutant and complemented FA-A or FA-G cells is statistically significant, as indicated by the asterisk (P = 0.001 and P = 0.006, respectively; unpaired t test). (E) HDR in Fanca–/– ES cell lines after I-SceI expression. The difference between the mutant and wild-type ES cells is statistically significant, as indicated by the asterisk (P = 0.0007 for clone C8 and P = 0.001 for clone H1).
Fig. 2.
Fig. 2.
FA patient-derived cell lines have normal levels of chromosomal NHEJ. (a) PCR strategy to detect DSB repair products at the DR-GFP locus. In addition to HDR, other DSB repair pathways are NHEJ and SSA, although neither of these results in GFP+ cells. All three DSB repair pathways result in I-SceI site loss; the homologous repair pathways HDR and SSA additionally result in replacement of the I-SceI site with a BcgI restriction site. (b) PCR products (primers F,R) from mutant and complemented FA-A and FA-G cell lines transfected with or without an I-SceI expression vector. Amplified products from the same reaction were digested with I-SceI or with I-SceI and BcgI. Also shown is a control PCR product obtained from a HEK-293 DR-GFP cell line that had been transfected with an I-SceI expression vector and enriched by flow cytometry for GFP+ cells. As expected, the amplified product is digestible with BcgI but not I-SceI. (c) Quantification of the PCR results from cells transfected with the I-SceI expression vector demonstrating comparable NHEJ repair in FA mutant and complemented cells. The mutant FA-A and FA-G cells have a lower amount of total repair product (uncut by I-SceI) than that found for the complemented cells (P = 0.0281 and P = 0.0234), but the NHEJ repair product (uncut by I-SceI and BcgI) is similar for the mutant and complemented cells. The percentage uncut is the ratio of the 0.65-kb band to the 0.5-kb band. For both the FA-A and FA-G complemented cell lines, but not the mutant cells, there is statistical significance between the level of total repair product and the NHEJ repair product (asterisk), indicating a significant contribution of the homologous repair product (i.e., cleavable by BcgI) to total repair (FA-A, P = 0.0174; FA-G, P = 0.0172).
Fig. 3.
Fig. 3.
FA patient-derived cell lines have reduced levels of DSB repair by SSA. (a) PCR strategy to quantify SSA. The 0.8-kb PCR fragment derived from primers SA-F and SA-R2 specifically detects the SSA repair product, whereas the 1.1-kb PCR fragment from primers SA-F and SA-R1 detects a structurally intact reporter, i.e., from HDR and NHEJ, as well as the parental DR-GFP reporter that retains the I-SceI site. (b) PCR products from mutant and complemented FA-A and FA-G cell lines transfected with or without an I-SceI expression vector. As a comparison, a similar analysis was performed on Brca2-deficient and control cells. Amplified products from parallel PCR reactions using primer sets SA-F/SA-R1 and SA-F/SA-R2 are shown. (c) Quantification of PCR results from mutant and complemented cells transfected with the I-SceI expression vector. Whereas the Brca2 mutant cells have an increased percentage of SSA relative to Brca2 wild-type cells, the FA-A and FA-G cell lines have a decreased percentage of SSA relative to their complemented counterparts. The level of SSA is derived from the ratio of the 0.8-kb band to the 1.1-kb band; because these bands are derived from independent PCR reactions, this is an arbitrary percentage and does necessarily not reflect the absolute level of SSA. For both the FA-A and FA-G cells, the difference between the amount of SSA for the mutant and complemented cells is statistically significant, as indicated by the asterisk (P = 0.0194 and P = 0.0086, respectively).
Fig. 4.
Fig. 4.
FA-D2 mutant cells have impaired homologous repair. (a) HDR in FA-D2 cells (PD20) expressing wild-type or mutant FANCD2 proteins. HDR is expressed relative to the mutant FA-D2 cells. Cells expressing wild-type FANCD2 or the ATM phosphorylation site mutant FANCD2-S222A have a higher level of HDR than cells expressing the monoubiquitination site mutant FANCD2-K561R or mutant (vector) cells. The difference between the mutant and FANCD2-complemented FA-D2 cells is statistically significant (asterisk, P = 0.0069), as is the difference between mutant and FANCD2-S222A-expressing cells (P = 0.046), using a paired t test. (b) FA-D2 cells have normal levels of chromosomal NHEJ. See Fig. 2a for a description of the primers. Note that the ratio of the 0.65-kb band to the 0.5-kb band after I-SceI/BcgI digestion is similar in intensity for each of the mutant or wild-type-complemented PD20 cells, indicating normal levels of NHEJ in the FA-D2 cells. (c and d) FA-D2 cells have reduced SSA. As with HDR, FA-D2 cells complemented with wild-type or FANCD2-S222A have higher levels of SSA than FANCD2-K561R-expressing or mutant FA-D2 cells. PCR products are derived from the strategy shown in Fig. 3a. As with the FA-A and FA-G cells, the difference in SSA levels is ≈2-fold between mutant and complemented FA-D2 cells. The difference between the mutant and FANCD2 or FANCD2-S222A complemented FA-D2 cells is statistically significant (P = 0.0076 and P = 0.0051, respectively).
Fig. 5.
Fig. 5.
Both upstream and downstream components of the FA pathway promote homologous repair by HDR and SSA, similar to BRCA1 but in contrast to BRCA2, which promotes HDR but suppresses SSA. See Discussion for further details.
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