BRCA1-independent ubiquitination of FANCD2

Monoubiquitination of the FANCD2 protein is a key step in the Fanconi anemia (FA) tumor suppressor pathway, coinciding with this molecule's accumulation at sites of genome damage. Strong circumstantial evidence points to a requirement for the BRCA1 gene product in this step. Here, we show that the purified BRCA1/BARD1 complex, together with E1 and UbcH5a, is sufficient to reconstitute the monoubiquitination of FANCD2 in vitro. Although siRNA-mediated knockdown of BRCA1 in human cells results in defective targeting of FANCD2 to sites of DNA damage, it does not lead to a defect in FANCD2 ubiquitination. Furthermore, ablation of the RING finger domains of either BRCA1 or BARD1 in the chicken B cell line DT40 also leaves FANCD2 modification intact. Consequently, while BRCA1 affects the accumulation of FANCD2 at sites of DNA damage, BRCA1/BARD1 E3 ligase activity is not essential for the monoubiquitination of FANCD2.     

An Integrated in Silico Approach to Analyze the Involvement of Single Amino Acid Polymorphisms in FANCD1/BRCA2-PALB2 and FANCD1/BRCA2-RAD51 Complex

Fanconi anemia (FA) is an autosomal recessive human disease characterized by genomic instability and a marked increase in cancer risk. The importance of FANCD1 gene is manifested by the fact that deleterious amino acid substitutions were found to confer susceptibility to hereditary breast and ovarian cancers. Attaining experimental knowledge about the possible disease-associated substitutions is laborious and time consuming. The recent introduction of genome variation analyzing in silico tools have the capability to identify the deleterious variants in an efficient manner. In this study, we conducted in silico variation analysis of deleterious non-synonymous SNPs at both functional and structural level in the breast cancer and FA susceptibility gene BRCA2/FANCD1. To identify and characterize deleterious mutations in this study, five in silico tools based on two different prediction methods namely pathogenicity prediction (SIFT, PolyPhen, and PANTHER), and protein stability prediction (I-Mutant 2.0 and MuStab) were analyzed. Based on the deleterious scores that overlap in these in silico approaches, and the availability of three-dimensional structures, structure analysis was carried out with the major mutations that occurred in the native protein coded by FANCD1/BRCA2 gene. In this work, we report the results of the first molecular dynamics (MD) simulation study performed to analyze the structural level changes in time scale level with respect to the native and mutated protein complexes (G25R, W31C, W31R in FANCD1/BRCA2-PALB2, and F1524V, V1532F in FANCD1/BRCA2-RAD51). Analysis of the MD trajectories indicated that predicted deleterious variants alter the structural behavior of BRCA2-PALB2 and BRCA2-RAD51 protein complexes. In addition, statistical analysis was employed to test the significance of these in silico tool predictions. Based on these predictions, we conclude that the identification of disease-related SNPs by in silico methods, in combination with MD approach has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases. The methods reviewed here generated a considerable amount of valuable data, but also the need for further validation.     

Functional assays for BRCA1 and BRCA2

Genetic testing for the two major breast cancer susceptibility genes has now been available for several years with more than 70,000 people tested in the USA alone. While the current genetic testing identifies many sequence alterations there are problems with both sensitivity and specificity of the assay. In particular, the genetic testing is limited in its ability to determine which of the many missense mutations identified in BRCA1 and BRCA2 actually predispose to cancer and which are simply neutral alterations. Here we will focus on the limitations in test result interpretation and we will explore how biochemistry and cell biology can help to clarify these issues. Although we limit our discussion to genetic testing of BRCA1 and BRCA2, the problem is common to an expanding group of genes, including ATM and MSH2, in which germ-line missense mutations may also confer increased risk of cancer. Here we advocate the use of functional assays to complement genetic data in the analysis of unclassified missense mutations and propose a set of standards to conduct and interpret these assays.     

Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.     

FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.     

FANCL Replaces BRCA1 as the Likely Ubiquitin Ligase Responsible for FANCD2 Monoubiquitination

Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.     

Functional interplay between BRCA2/FancD1 and FancC in DNA repair

A rare hereditary disorder, Fanconi anemia (FA), is caused by mutations in an array of genes, which interact in a common FA pathway/network. These genes encode components of the FA "core" complex, a key factor FancD2, the familial breast cancer suppressor BRCA2/FancD1, and Brip1/FancJ helicase. Although BRCA2 is known to play a pivotal role in homologous recombination repair by regulating Rad51 recombinase, the precise functional relationship between BRCA2 and the other FA genes is unclear. Here we show that BRCA2-dependent chromatin loading of Rad51 after mitomycin C treatment was not compromised by disruption of FANCC or FANCD2. Rad51 and FancD2 form colocalizing subnuclear foci independently of each other. Furthermore, we created a conditional BRCA2 truncating mutation lacking the C-terminal conserved domain (CTD) (brca2DeltaCTD), and disrupted the FANCC gene in this background. The fancc/brca2DeltaCTD double mutant revealed an epistatic relationship between FANCC and BRCA2 CTD in terms of x-ray sensitivity. In contrast, levels of cisplatin sensitivity and mitomycin C-induced chromosomal aberrations were increased in fancc/brca2DeltaCTD cells relative to either single mutant. Taken together, these results indicate that FA proteins work together with BRCA2/Rad51-mediated homologous recombination in double strand break repair, whereas the FA pathway plays a role that is independent of the CTD of BRCA2 in interstrand cross-link repair. These results provide insights into the functional interplay between the classical FA pathway and BRCA2.     

FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination

Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.     

Haplotype analysis of eight genes of the monoubiquitinated FANCD2–DNA damage–repair pathway in breast cancer patients

Background: Ten genes are associated with increased susceptibility to inherited breast cancer have also been associated with population breast cancer risk, and all are involved directly or indirectly in the monoubiquitinated FANCD2–DNA damage repair pathway. We analyzed 13 haplotype blocks in eight of these genes to estimate the breast cancer risk conferred by individual haplotypes. Methods: Haplotype blocks were constructed with 48 tag single-nucleotide polymorphisms (tSNPs) identified in eight breast cancer susceptibility genes, TP53, PTEN, CHEK2, ATM, NBS1, RAD50, BRIP1, and PALB2. Genotyping was performed by SNPscan on 734 female patients and 672 female age-matched controls. Results: Forty-five tSNPs were successfully genotyped by SNPscan, and call rates for each tSNP were above 98.9%. Thirteen haplotype blocks of eight genes were constructed with 41 successfully genotyped tSNPs. We found that seven haplotypes from four haplotype blocks located within three genes (NBS1, PTEN, and BRIP1) were significantly associated with breast cancer risk. Among these, four haplotypes (ATC in block 1 of NBS1, GCCCC and GCCCT in block 2 of NBS1, and GCT in block 2 of BRIP1) were correlated with breast cancer risk in sporadic cases (OR (95% CI) 1.350(1.124–1.623), 0.752(0.584–0.969), 0.803(0.649–0.993), and 0.776(0.604–0.997), respectively), and only one haplotype (GGCCT in block 2 of NBS1) was significantly associated with breast cancer risk in familial and early-onset cases (OR(95% CI) 1.902(1.134–3.191)). Conclusions: Four haplotypes within two genes (NBS1 and BRIP1) involved in the monoubiquitinated FANCD2–DNA damage–repair pathway are significantly associated with increased sporadic breast cancer risk, while one haplotype within NBS1 is correlated with an increased risk of familial or early-onset breast cancer, indicating that specific haplotypes may be distinct predictors of breast cancer.     

FANCJ/BRIP1 recruitment and regulation of FANCD2 in DNA damage responses

FANCJ/BRIP1 encodes a helicase that has been implicated in the maintenance of genomic stability. Here, to better understand FANCJ function in DNA damage responses, we have examined the regulation of its cellular localization. FANCJ nuclear foci assemble spontaneously during S phase and are induced by various stresses. FANCJ foci colocalize with the replication fork following treatment with hydroxyurea, but not spontaneously. Using FANCJ mutants, we find that FANCJ helicase activity and the capacity to bind BRCA1 are both involved in FANCJ recruitment. Given similarities to the recruitment of another Fanconi anemia protein, FANCD2, we tested for colocalization of FANCJ and FANCD2. Importantly, these proteins show substantial colocalization, and FANCJ promotes the assembly of FANCD2 nuclear foci. This process is linked to the proper localization of FANCJ itself since both FANCJ and FANCD2 nuclear foci are compromised by FANCJ mutants that abrogate its helicase activity or interaction with BRCA1. Our results suggest that FANCJ is recruited in response to replication stress and that FANCJ/BRIP1 may serve to link FANCD2 to BRCA1.     

Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway

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Assessing Associations between the AURKA-HMMR-TPX2-TUBG1 Functional Module and Breast Cancer Risk in BRCA1/2 Mutation Carriers

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04 – 1.15, p = 1.9 x 10⁻⁴ (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03 – 1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted p_interaction values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients’ survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers.     

UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination

The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.     

BRCA1 and BRCA2 in hereditary breast cancer

The hereditary breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, have established roles in genome integrity maintenance and in the control of homologous recombination. Recent work has produced valuable insights into the mechanisms of action of the gene products. This review summarizes some of these advances, and attempts to place them in the context of known functions of the genes.     

Screening Fanconi anemia lymphoid cell lines of non-A, C, D2, E, F, G subtypes for defects in BRCA2/FANCD1

Biallelic mutations in BRCA2/FANCD1 were recently recognized as a rare cause of Fanconi anemia (FA). Using immunodetection with an antiserum directed against the carboxyterminus of the BRCA2 protein, we screened 38 lymphoid cell lines from FA patients whom we could not previously assign, via retroviral complementation analysis, to any of six known FA complementation groups (FA-A, -C, -D2, -E, -F, or -G). Three of these 38 cell lines lacked the 380-kDa BRCA2 signal on immunoblots. DNA sequencing showed biallelic compound and truncating mutations in two of the immuno-negative cell lines, whereas a monoallelic frameshift mutation and an amino acid substitution were detected in the third cell line. Our data show that less than 10% of unassigned FA cell lines harbor truncating mutations in BRCA2/FANCD1. This finding strongly suggests the existence of (an) additional, as yet unknown FA gene(s).     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

New complexities for BRCA1 and BRCA2

A large number of diverse functions have been attributed to the BRCA1 and BRCA2 breast cancer susceptibility genes. Here we review recent progress in the field.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.