ALBERT

Description: Fanconi anemia (FA) is a genetic disease marked by bone marrow failure, congenital defects, and cancer. In spite of the identification of at least 8 genes, the biochemistry of the disease and its normal pathway in the cell remains elusive. The FA core complex is composed of at least 5 proteins, 2 of which, FANCA and FANCG, we have shown to be phosphorylated. In these studies, we show that both FANCA and FANCG are phosphorylated in response to DNA damage. In the case of FANCG, we have mapped the site of this phosphorylation to serine 7, using a phosphoserine 7 FANCG antiserum. Because of the link of FA function and the FA core complex-dependent monoubiquitination that occurs both as a result of DNA damage as well as at S phase, we also examined if phosphorylation occurred at S phase as well. While FANCG serine 7 phosphorylation occurs both at S phase and after DNA damage (similar to FANCD2 monoubiquitination), FANCA phosphorylation occurs only after DNA damage. Recent data have implicated the kinase ATR as important in the pathway. In order to assess whether a downstream target of ATR is differentially phosphorylated in FA cells, we tested the phosphorylation status of chk1 in FA-A mutant and corrected cells. Chk1 kinase is phosphorylated at serine 318 in response to DNA damage only in corrected cells but not mutant FA cells, while signaling through chk2 kinase is unaffected. These data suggest the importance of phosphorylation in the FA pathway in the regulation of both cellular responses to DNA damage as well as engagement of the cell cycle.

Description: Fanconi anemia (FA) is an inherited hematological disorder characterized by birth defects, bone marrow failure, and cancer susceptibility. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to cross linking reagents such as mitomycin C (MMC). Cloned FA genes cooperate in a signaling pathway activated by DNA damage during replication, and monoubiquitination of FANCD2 is the critical downstream event. The check point kinase ATR is required for the efficient monoubiquitination of FANCD2. Inducible ATR-dependent phosphorylation of threonine 691 and serine 717 are reported to enhance the monoubiquitination of FANCD2 and resistance to MMC. In the present study, we have devised means to purify the FANCD2 protein into multiple subcomplexes. In doing so, we have identified at least 3 distinct subcomplexes: a pure multimeric precursor form comprised of FANCD2 polypeptides, a 1 MD form that is chromatin bound, and a 2 MD form that is in the nucleosol. Electron microscopic analysis of the precursor form reveals homogenous appearing complexes. Mass spectroscopic analysis of the 1MD FANCD2 complex has identified the FANCI protein, among others, which confirms recent reports of FANCD2-FANCI interaction. Further mass spectroscopic analysis of FANCD2 has identified a novel phosphorylation site on FANCD2, serine 331, which is further confirmed by immunoblot using phosphospecific antibody against phospho-serine 331. This phosphorylation site is conserved in other species, including mouse, rat, xenopus, and tetraodon nigroviridis. Single amino acid mutation by alanine at this site results in a complete loss of monoubquitination of FANCD2 and resistance to MMC, suggesting that the phosphorylation of S331 is a functionally significant event. Moreover, this site fits the consensus sequence for a CHK1 substrate, which in turn is a substrate of target for CHK1, a substrate of ATR, which is also required for monoubiquitination of FANCD2. These findings further support the correlation between phosphorylation and monoubiquitination of FANCD2 in the DNA damage response with the intervention of CHK1 as a mediator of ATR and suggest dynamic regulation of FANCD2 subcomplexes within the FA pathway.

Description: Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway.

Description: Fanconi anemia (FA) represents a paradigm of rare genetic diseases, where the quest for cause and cure has led to seminal discoveries in cancer biology. Although a total of 16 FA genes have been identified thus far, the biochemical function of many of the FA proteins remains to be elucidated. FA is rare, yet the fact that 5 FA genes are in fact familial breast cancer genes and FA gene mutations are found frequently in sporadic cancers suggest wider applicability in hematopoiesis and oncology. Establishing the interaction network involving the FA proteins and their associated partners has revealed an intersection of FA with several DNA repair pathways, including homologous recombination, DNA mismatch repair, nucleotide excision repair, and translesion DNA synthesis. Importantly, recent studies have shown a major involvement of the FA pathway in the tolerance of reactive aldehydes. Moreover, despite improved outcomes in stem cell transplantation in the treatment of FA, many challenges remain in patient care.

Description: FANCD2 is a key player in Fanconi anemia (FA) pathway. It has been shown that PCNA and Rad18 can interact with FANCD2 and regulate FANCD2 monoubiquitination. Rad18 is the E3 ubiquitin ligase responsible for PCNA monoubiquitination, which is critical for PCNA function in translesion synthesis (TLS). Previous work by us and others has shown that the FA pathway interacts with PCNA and RAD18. Even though FA cells demonstrate unequivocal sensitivity to crosslinkers such as mitomycin C (MMC), we find surprisingly that they are largely resistant to hydroxyurea (HU), except for cells containing no expression of FANCD2. In order to understand the mechanism for this finding, we explored the interaction of FANCD2, Rad51, Rad18, and PCNA, which form a complex and is enhanced upon HU exposure. While FANCD2 is required, monoubiquitination of FANCD2 is not. In addition, PCNA monoubiqutination follows exactly the same pattern. The monoubiquitination of PCNA in response to HU in FANCA deficient mutant confirms that non-ubiquitinated FANCD2 can still support PCNA monoubiquitination. Rad51, but not BRCA1 is also required in regulating PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion polymerase Pol H chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knock down cells and Rad51 siRNA knock down cells. Our data suggest that FANCD2 and Rad51 play an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with mitomycin C (MMC) treatment, indicating that DNA repair systems differentially respond to different types of DNA damage. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2377 FANCD2 is a key player in FA pathway. It has been shown that FANCD2 can interact with PCNA and with Rad18, the ubiquitin ligase responsible for PCNA mono-ubiquitination. The mono-ubiquitination of PCNA is very important for its function in translesion synthesis. We found that in response to DNA damage agent hyxdroxyurea (HU) the interaction of FANCD2 with Rad18 or Rad51, and the interaction of Rad51 with Rad18 or PCNA was enhanced. FANCD2 is required for increased interaction between Rad51 and Rad18 indicating that FANCD2, Rad51 and Rad18 form a complex in response to HU. Rad18 was required for PCNA mono-ubiquitination in response to HU. FANCD2 deficient cells failed to enhance the interaction between Rad18 and Rad51. Furthermore, PCNA mono-ubiquitination was impaired in FANCD2 deficient cells in response to HU. FANCD2 mono-ubiquitination deficient mutant partially rescued PCNA mono-ubiquitination. The partial mono-ubiquitination of PCNA in response to HU in FANCA deficient mutant confirmed the role of non-ubiquitinated FANCD2 in PCNA mono-ubiquitination. The normal mono-ubiquitination of PCNA in FANCJ deficient mutant confirmed that the effect of FANCD2 in PCNA mon-ubiquitination is not due to FA pathway deficiency. Rad51 was also involved in regulating PCNA mono-ubiquitination in response to HU. Rad51 siRNA knock down cells showed decreased PCNA mono-ubiquitination in response to HU. The role of Rad51 in regulating PCNA mono-ubiquitination did not require BRCA1, indicating that this function is independent of HR. More importantly FANCD2 deficient cells were hypersensitive to HU, whereas FANCD2 mono-ubiquitination deficient mutant cells, FANCD2 corrected cells, FANCA deficient cells and FANCJ deficient cells were not hypersensitive to HU. Our data indicate that FANCD2 plays an important role in PCNA mono-ubiquitination and translesion synthesis partially in a mono-ubiquitination independent manner. Rad51 also plays an important role in PCNA mono-ubiquitination and translesion synthesis in a HR independent fashion. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1095 Poster Board I-117 Introduction Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). Central to this pathway is the monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI. Recent reports implicate mismatch repair factors in the repair of ICLs and have shown that FANCJ interacts with the MutLαa complex. Here we show that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Methods Cell lines used: HeLa, human lung carcinoma cell line H1299, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agent mitomycin C (MMC). Immunoprecipitation was used to demonstrate the interaction between FANCD2 and MSH2, MLH1, and MSH3. Survival assays were performed by crystal violet staining and extraction. Chromatin loading of FANCD2 and FANCI was determined by cellular fractionation and western blot. Results Through chromatographic purification of FANCD2-containing protein complexes, we identified MSH2 and MLH1 as FANCD2-interacting proteins. Immunoprecipitation using HeLa cell extracts confirmed the interaction between FANCD2, MSH2, MSH3, and MLH1 in vivo. These interactions are all induced upon damage with a DNA crosslinking agent and MSH2 specifically interacts only with the monoubiquitylated form of FANCD2. Additionally, the FANCD2-MSH2 interaction requires ATR, but not ATM, BRCA1, MSH3, or ERCC1/XPF. Human cells lacking MSH2 display increased sensitivity to mitomycin C as compared to their corrected counterparts. FANCD2 and FANCI monoubiquitylation is also greatly diminished in these cells, while cells lacking MLH1 show no effect. Cellular fractionation of MSH2-deficient cells shows that FANCD2 and FANCI are not efficiently loaded onto chromatin after treatment with DNA-damaging agents, while MLH1-deficient cells again show no effect. Interestingly, while knockdown of either MSH2 or FANCD2 in H1299 cells results in increased sensitivity to MMC, double knockdown of both proteins corrects this sensitivity on par with controls. oConclusions These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway, likely through recognition of the DNA lesion. Understanding this role could lead to the development of new therapies for the treatment of patients both with FA and cancer. Disclosures No relevant conflicts of interest to declare.