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Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway.

ABSTRACT: Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the ~300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

SUBMITTER: Joo W

PROVIDER: S-EPMC3310437 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway.

Joo Woo W Xu Guozhou G Persky Nicole S NS Smogorzewska Agata A Rudge Derek G DG Buzovetsky Olga O Elledge Stephen J SJ Pavletich Nikola P NP

Science (New York, N.Y.) 20110701 6040

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the ~300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeri ...[more]

PMID: 21764741

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Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage.

Project description:Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.

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Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.

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Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase.

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Project description:Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes.

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Dysfunctional DNA repair pathway via defective FANCD2 gene engenders multifarious exomic and transcriptomic effects in Fanconi anemia.

Project description:BACKGROUND:Fanconi anemia (FA) affects only one in 130,000 births, but has severe and diverse clinical consequences. It has been theorized that defects in the FA DNA cross-link repair complex lead to a spectrum of variants that are responsible for those diverse clinical phenotypes. METHODS:Using NextGen sequencing, we show that a clinically derived FA cell line had accumulated numerous genetic variants, including high-impact mutations, such as deletion of start codons, introduction of premature stop codons, missense mutations, and INDELs. RESULTS:About 65% of SNPs and 55% of INDELs were found to be commonly present in both the FA dysfunctional and retrovirally corrected cell lines, showing their common origin. The number of INDELs, but not SNPs, is decreased in FANCD2-corrected samples, suggesting that FANCD2 deficiency preferentially promotes the origin of INDELs. These genetic modifications had a considerable effect on the transcriptome, with statistically significant changes in the expression of 270 genes. These genetic and transcriptomic variants significantly impacted pathways and molecular functions, spanning a diverse spectrum of disease phenotypes/symptoms, consistent with the disease diversity seen in FA patients. CONCLUSION:These results underscore the consequences of defects in the DNA cross-link repair mechanism and indicate that accumulating diverse mutations from individual parent cells may make it difficult to anticipate the longitudinal clinical behavior of emerging disease states in an individual with FA.

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The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

Project description:Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

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Fanconi anemia pathway regulation by FANCI in prostate cancer.

Project description:Prostate cancer is one of the leading causes of death among men worldwide, and thus, research on the genetic factors enabling the formation of treatment-resistant cancer cells is crucial for improving patient outcomes. Here, we report a cell line-specific dependence on FANCI and related signaling pathways to counteract the effects of DNA-damaging chemotherapy in prostate cancer. Our results reveal that FANCI depletion results in significant downregulation of Fanconi anemia (FA) pathway members in prostate cancer cells, indicating that FANCI is an important regulator of the FA pathway. Furthermore, we found that FANCI silencing reduces proliferation in p53-expressing prostate cancer cells. This extends the evidence that inactivation of FANCI may convert cancer cells from a resistant state to an eradicable state under the stress of DNA-damaging chemotherapy. Our results also indicate that high expression of FA pathway genes correlates with poorer survival in prostate cancer patients. Moreover, genomic alterations of FA pathway members are prevalent in prostate adenocarcinoma patients; mutation and copy number information for the FA pathway genes in seven patient cohorts (N = 1,732 total tumor samples) reveals that 1,025 (59.2%) tumor samples have an alteration in at least one of the FA pathway genes, suggesting that genomic alteration of the pathway is a prominent feature in patients with the disease.

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Fanconi Anemia pathway regulation by FANCI in prostate cancer

Project description:Prostate cancer is one of the leading causes of death among men worldwide and thus research on the genetic factors enabling the formation of treatment resistant cancer cells is crucial for improving patient outcomes.Here, we report a cell-line specific dependence on FANCI and related signalling pathways to counteract effects of DNA damaging chemotherapy in prostate cancer. Our results reveal that FANCI depletion results in significant downregulation of Fanconi anemia (FA) pathway members in prostate cancer cells indicating that FANCI is an important regulator of FA pathway. Furthermore, we found that FANCI silencing reduces proliferation in p53-expressing prostate cancer cells. This extends the evidence that inactivation of FANCI may convert cancer cells from a resistant to an eradicable state under the stress of DNA-damaging chemotherapy. Our results also indicate that high expression of FA pathway genes correlates with poorer survival in prostate cancer patients. Moreover, genomic alterations of FA pathway members are prevalent in prostate adenocarcinoma patients; mutation and copy number information for the FA pathway genes in seven patient cohorts (N = 1732 total tumor samples) reveals that 1025 (59.2%) tumor samples have an alteration in at least one of the FA pathway genes, suggesting that genomic alteration of the pathway is a prominent feature in patients with the disease.

2023-09-29 | GSE211363 | GEO

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