Dataset Information

Structure of the Fanconi anaemia monoubiquitin ligase complex

ABSTRACT: The Fanconi Anemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks. Genetic inactivation of this pathway impairs development, prevents blood production and promotes cancer. The key molecular step in the FA pathway is the monoubiquitination of a heterodimer of FANCI-FANCD2 by the FA core complex - a megadalton multiprotein E3 ubiquitin ligase. Monoubiquitinated FANCI-FANCD2 then activates a pathway to remove the DNA crosslink. Lack of molecular insight into the FA core complex limits a detailed explanation of how this vital DNA repair pathway functions. Here we reconstituted an active, recombinant FA core complex, and used electron cryo-microscopy (cryo-EM) and mass spectrometry to determine its overall structure. The FA core complex is comprised of a central symmetric dimer of the FANCB and FAAP100 subunits, flanked by two copies of the RING finger protein, FANCL. This acts as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. The two FANCL subunits are positioned at opposite ends of the complex in an unusual asymmetric arrangement, distinct from other E3 ligases. We propose that each of the two FANCL subunits play unique roles within the complex – one is a structural component while the other monoubiquitinates FANCD2. The cryo-EM structure of the FA core complex, supported by crosslinking mass spectrometry and native mass spectrometry, therefore provides a foundation for a detailed understanding of this fundamental DNA repair pathway.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Gallus Gallus (chicken)

SUBMITTER: Francis O'Reilly

LAB HEAD: Juri Rappsilber

PROVIDER: PXD014282 | Pride | 2019-10-16

REPOSITORIES: Pride

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			Action	DRS
	B181122_28_Lumos_FO_IN_120_FAcomplex_BS3_SEC6_rep1.raw	Raw
	B181122_29_Lumos_FO_IN_120_FAcomplex_BS3_SEC6_rep2.raw	Raw
	B181122_30_Lumos_FO_IN_120_FAcomplex_BS3_SEC7_rep1.raw	Raw
	B181122_31_Lumos_FO_IN_120_FAcomplex_BS3_SEC7_rep2.raw	Raw
	B181122_32_Lumos_FO_IN_120_FAcomplex_BS3_SEC8_rep1.raw	Raw

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Structure of the Fanconi anaemia monoubiquitin ligase complex.

Shakeel Shabih S Rajendra Eeson E Alcón Pablo P O'Reilly Francis F Chorev Dror S DS Maslen Sarah S Degliesposti Gianluca G Russo Christopher J CJ He Shaoda S Hill Chris H CH Skehel J Mark JM Scheres Sjors H W SHW Patel Ketan J KJ Rappsilber Juri J Robinson Carol V CV Passmore Lori A LA

Nature 20191030 7781

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core c ...[more]

PMID: 31666700

Similar Datasets

Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Project description:Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure and increased susceptibility to cancer. Of the fifteen FA proteins, Fanconi anemia group C (FANCC) is one of eight FA core complex components of the FA pathway. Unlike other FA core complex proteins, FANCC is mainly localized in the cytoplasm, where it is thought to function in apoptosis, redox regulation, cytokine signaling and other processes. Previously, we showed that regulation of FANCC involved proteolytic processing during apoptosis. To elucidate the biological significance of this proteolytic modification, we searched for molecular interacting partners of proteolytic FANCC fragments. Among the candidates obtained, the transcriptional corepressor protein C-terminal binding protein-1 (CtBP1) interacted directly with FANCC and other FA core complex proteins. Although not required for stability of the FA core complex or ubiquitin ligase activity, CtBP1 is essential for proliferation, cell survival and maintenance of chromosomal integrity. Expression profiling of CtBP1-depleted and FA-depleted cells revealed that several genes were commonly up- and down-regulated, including the Wnt antagonist Dickkopf-1 (DKK1). These findings suggest that FA and Wnt signaling via CtBP1 could share common effectors. HeLa cell line was grown in DMEM media supplemented with 10% FBS and were incubated in 5% CO2 at 37Â°C. A four-plasmid (pRSV-Rev, pMDLg/pRRE, pMD2.G and pLKO.1) expression system was used for lentiviral production. Different pLKO.1 plasmids carrying shRNAs targeting FANCA, FANCD2, CtBP1 or CtBP2 or a lentiviral control vector pLKO.1-scrambled were used. Lentiviral particles were produced via calcium phosphate-mediated transient transfection of the four plasmids into HEK293T cells. Cells were exposed to appropriate lentiviral particles. In each experiment, HeLa cells were transduced for 6 hours with filtered supernatant containing recombinant lentiviral particles. After transduction, the cells were cultured for 72 hours. Total RNA extracts from 3 different samples of each scrambled, CtBPs and FANCD2 shRNA treated HeLa cells were subjected to gene expression profiling via microarray analysis. Gene expression profiles were determined with Affymetrix GeneChipÂ® Human Gene 1.0.