scholarly journals Towards a Molecular Understanding of the Fanconi Anemia Core Complex

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Charlotte Hodson ◽  
Helen Walden
Keyword(s):  
Fanconi Anemia ◽  
Molecular Mechanisms ◽  
Genome Instability ◽  
Genetic Disorder ◽  
Single Gene ◽  
Main Function ◽  
Core Complex ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks ◽  
Interstrand Crosslinking

Fanconi Anemia (FA) is a genetic disorder characterized by the inability of patient cells to repair DNA damage caused by interstrand crosslinking agents. There are currently 14 verified FA genes, where mutation of any single gene prevents repair of DNA interstrand crosslinks (ICLs). The accumulation of ICL damage results in genome instability and patients having a high predisposition to cancers. The key event of the FA pathway is dependent on an eight-protein core complex (CC), required for the monoubiquitination of each member of the FANCD2-FANCI complex. Interestingly, the majority of patient mutations reside in the CC. The molecular mechanisms underlying the requirement for such a large complex to carry out a monoubiquitination event remain a mystery. This paper documents the extensive efforts of researchers so far to understand the molecular roles of the CC proteins with regard to its main function in the FA pathway, the monoubiquitination of FANCD2 and FANCI.

Nuclear Localization of Fanconi Anemia Protein FANCA Is Regulated by Hsc70/Hsp90 Chaperone Machinery.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2835-2835
Author(s):  
Tsukasa Oda ◽  
Hidenobu Miyaso ◽  
Takayuki Yamashita
Keyword(s):  
Nuclear Localization ◽  
Fanconi Anemia ◽  
Subcellular Distribution ◽  
Molecular Mechanisms ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Specific Inhibitor ◽  
Current Evidence ◽  
Core Complex ◽  
Hsp90 Chaperone

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, cancer susceptibility and cellular hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). Current evidence indicates that formation of a nuclear multiprotein complex (core complex) including six FA proteins FANCA/C/E/F/G/L is essential for FANCL/PHF9 ubiquitin ligase-mediated activation of FANCD2 into a monoubiquinated form, which participates in BRCA1 and FANCD1/BRCA2-mediated DNA repair (the FA/BRCA pathway). Subcellular distribution of FANCA plays a crucial role in the regulation of the FA/BRCA pathway. However, the underlying molecular mechanisms are not fully understood. To address this issue, we tried to identify FANCA-associated proteins. To this end, Flag-FANCA ectopically expressed in HeLa cells was immunopurified from the cytoplasmic fraction, using anti-Flag antibody-conjugated sepharose beads. Analysis of the immune complex on SDS polyacrylamide gel electrophoresis revealed that several proteins of Mr. 60–70 kD specifically associated with Flag-FANCA. These proteins were identified as FANCG and Hsc (heat shock cognate protein) 70 by LC-MS/MS. Immunoblot analysis showed that FANCA associated with Hsp90 as well as Hsc70. Hsc70 is an ATP-dependent molecular chaperone highly homologous to Hsp70 and often cooperates with Hsp90 to form a chaperone machinery involved in the regulation of diverse protein functions. Patient-derived FANCA mutants failed to bind FANCC but associated with larger amounts of Hsc70 than wt-FANCA, indicating that the interaction between FANCA and Hsc70 is not mediated by FANCC, as suggested by previous observations of the interaction of FANCC with Hsp70. To study the role of Hsc70 and Hsp90 in the regulation of FANCA, we examined effects of a dominant-negative (dn) form of Hsc70 with inactivated ATPase activity, and a specific inhibitor of Hsp90, 17-AAG (a geldanamycin analog). Overexpression of dn-Hsc70 inhibited nuclear localization of FANCA and inhibited its core complex formation, whereas wt-Hsc70 did not. 17-AAG induced cytoplasmic distribution and proteosomal degradation of FANCA and suppressed FANCD2 mono-ubiquitination. Taken together, these results suggest that Hsc70/Hsp90 chaperone machinery interacts with FANCA and regulates its subcellular distribution and stability, thereby controlling activation of the FA/BRCA pathway.

scholarly journals Structure of the Fanconi Anemia Core–UBE2T complex poised to ubiquitinate bound FANCI–FANCD2

2019 ◽  
Author(s):  
Shengliu Wang ◽  
Renjing Wang ◽  
Christopher Peralta ◽  
Ayat Yaseen ◽  
Nikola P. Pavletich
Keyword(s):  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Single Copy ◽  
Core Complex ◽  
Interstrand Crosslinks ◽  
Ubiquitination Site ◽  
Pathway Activation ◽  
Dna Interstrand Crosslinks ◽  
Ubiquitin Conjugating Enzyme

ABSTRACTThe Fanconi Anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks (ICLs). The pathway is activated when a replication fork stalls because of an ICL or other replication stress. A central event in pathway activation is the mono-ubiquitination of the FANCI-FANCD2 (ID) complex by the FA Core complex, a ubiquitin ligase of nine subunits. Here we describe the cryo-EM structures of the 1.1 MDa FA Core at 3.1 angstroms, except for the FANCA subunit at 3.4, and of the complex containing Core, ID and the UBE2T ubiquitin conjugating enzyme at 4.2 angstroms. The Core has unusual stoichiometry with two copies of FANCB, FAAP100, FANCA, FAAP20, FANCG, FANCL, but only a single copy of FANCC, FANCE and FANCF. This is due to homodimers of FANCA and FANCB having incompatible 2-fold symmetry, resulting in an overall asymmetric assembly of the other subunits. The asymmetry is crucial, as it prevents the binding of a second FANC-C-E-F sub-complex that inhibits UBE2T recruitment by FANCL, and instead creates an ID binding site. The single active FANCL-UBE2T binds next to the FANCD2 ubiquitination site, prying open the FANCI-FANCD2 interface within which the ubiquitination sites are buried. These structures and biochemical data indicate a single active site ubiquitinates FANCD2 and FANCI sequentially, shedding light on a central event in the FA pathway.

scholarly journals Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA

2020 ◽  
Author(s):  
Martin L. Rennie ◽  
Kimon Lemonidis ◽  
Connor Arkinson ◽  
Viduth K. Chaugule ◽  
Mairi Clarke ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Large Scale ◽  
The Other ◽  
Molecular Function ◽  
Post Translational Modifications ◽  
Hydrophobic Residues ◽  
Interstrand Crosslinks ◽  
Double Stranded Dna ◽  
Dna Interstrand Crosslinks ◽  
Lysine Residues

AbstractThe Fanconi Anemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks, and which is additionally activated in response to other forms of replication stress. A key step in the activation of the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here we find that ubiquitination of FANCD2 acts to increase ID2’s affinity for double stranded DNA via promoting/stabilizing a large-scale conformational change in the complex, resulting in a secondary “Arm” ID2 interphase encircling DNA. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1/UAF deubiquitination, with key hydrophobic residues of FANCI’s ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilise a conformation in which the ID2 complex encircles DNA.

scholarly journals UHRF1 Is a Sensor for DNA Interstrand Crosslinks and Recruits FANCD2 to Initiate the Fanconi Anemia Pathway

Cell Reports ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. 1947-1956 ◽  
Author(s):  
Chih-Chao Liang ◽  
Bao Zhan ◽  
Yasunaga Yoshikawa ◽  
Wilhelm Haas ◽  
Steven P. Gygi ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Fanconi Anemia Pathway ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks

scholarly journals Polygenic mutations model the pleiotropic disease of Fanconi Anemia

2020 ◽  
Author(s):  
Karl-Heinz Tomaszowski ◽  
Sunetra Roy ◽  
Caezaan Keshvani ◽  
Martina Ott ◽  
Courtney DiNardo ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Human Disease ◽  
Genome Instability ◽  
Bone Marrow Failure ◽  
Single Gene ◽  
Cancer Therapeutics ◽  
Epistasis Analysis ◽  
Whole Exome ◽  
Frequent Event ◽  
Cellular Sensitivity

AbstractFanconi Anemia (FA) is a prototypic genetic disease signified by heterogeneous phenotypes including cancer, bone marrow failure, short stature, congenital abnormalities, infertility, sub-mendelian birth rate, genome instability and high cellular sensitivity to cancer therapeutics1-4. Clinical diagnosis is confirmed by identifying biallelic, homo- or hemizygous mutations in any one of twenty-three FANC genes1,5. Puzzlingly, inactivation of one single Fanc gene in mice fails to faithfully model the human disease manifestations6-8. We here delineate a preclinical Fanc mouse model with mutations in two genes, Fancd1/Brca2 and Fanco/Rad51c, that recapitulates the severity and heterogeneity of the human disease manifestations including death by cancer at young age. Surprisingly, these grave phenotypes cannot be explained by the sum of phenotypes seen in mice with single gene inactivation, which are unremarkable. In contrast to expectations from classic epistasis analysis of genetic pathways, the data instead reveal an unexpected functional synergism of polygenic Fanc mutations. Importantly in humans, whole exome sequencing uncovers that FANC co-mutation in addition to the identified inactivating FANC gene mutation is a frequent event in FA patients. Collectively, the data establish a concept of polygenic stress as an important contributor to disease manifestations, with implications for molecular diagnostics.

scholarly journals DNA clamp function of the mono-ubiquitinated Fanconi Anemia FANCI-FANCD2 complex

2019 ◽  
Author(s):  
Renjing Wang ◽  
Shengliu Wang ◽  
Ankita Dhar ◽  
Christopher Peralta ◽  
Nikola P. Pavletich
Keyword(s):  
Fanconi Anemia ◽  
Translesion Synthesis ◽  
The Other ◽  
Replication Forks ◽  
Dna Structures ◽  
Cancer Predisposition Syndrome ◽  
Interstrand Crosslinks ◽  
Branched Dna ◽  
Closed Ring ◽  
Dna Interstrand Crosslinks

ABSTRACTThe FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer predisposition syndrome, is required for the repair of replication forks stalled at DNA interstrand crosslinks (ICL) and related lesions1. The FA pathway is activated when two replication forks converge onto an ICL2, triggering the mono-ubiquitination of the ID complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion synthesis and homologous recombination, but its function was hitherto unknown1,3. Here, the 3.48 Å cryo-EM structure of mono-ubiquitinated ID (IDUb) bound to DNA reveals that it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. The structures, in conjunction with biochemical data, indicate the mono-ubiquitinated ID complex looses its preference for ICL and related branched DNA structures, becoming a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how mono-ubiquitination in general can induce an alternate structure with a new function.

scholarly journals Coordinated Cut and Bypass: Replication of Interstrand Crosslink-Containing DNA

2021 ◽  
Vol 9 ◽  
Author(s):  
Qiuzhen Li ◽  
Kata Dudás ◽  
Gabriella Tick ◽  
Lajos Haracska
Keyword(s):  
Dna Replication ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Defense Mechanism ◽  
Genome Instability ◽  
Dna Lesions ◽  
Fanconi Anemia Pathway ◽  
Interstrand Crosslinks ◽  
Dna Lesion ◽  
Interstrand Crosslink

DNA interstrand crosslinks (ICLs) are covalently bound DNA lesions, which are commonly induced by chemotherapeutic drugs, such as cisplatin and mitomycin C or endogenous byproducts of metabolic processes. This type of DNA lesion can block ongoing RNA transcription and DNA replication and thus cause genome instability and cancer. Several cellular defense mechanism, such as the Fanconi anemia pathway have developed to ensure accurate repair and DNA replication when ICLs are present. Various structure-specific nucleases and translesion synthesis (TLS) polymerases have come into focus in relation to ICL bypass. Current models propose that a structure-specific nuclease incision is needed to unhook the ICL from the replication fork, followed by the activity of a low-fidelity TLS polymerase enabling replication through the unhooked ICL adduct. This review focuses on how, in parallel with the Fanconi anemia pathway, PCNA interactions and ICL-induced PCNA ubiquitylation regulate the recruitment, substrate specificity, activity, and coordinated action of certain nucleases and TLS polymerases in the execution of stalled replication fork rescue via ICL bypass.

scholarly journals Structural basis of FANCD2 deubiquitination by USP1-UAF1

2020 ◽  
Author(s):  
Martin L. Rennie ◽  
Connor Arkinson ◽  
Viduth K. Chaugule ◽  
Rachel Toth ◽  
Helen Walden
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Conformational Changes ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Interstrand Crosslinks ◽  
Biochemical Assays ◽  
Dna Interstrand Crosslinks ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

AbstractUbiquitin-Specific Protease 1 (USP1), together with the cofactor UAF1, acts during DNA repair processes to specifically to remove mono-ubiquitin signals. The mono-ubiquitinated FANCI-FANCD2 heterodimer is one such substrate and is involved in the repair of DNA interstrand crosslinks via the Fanconi Anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin, and bound to mono-ubiquitinated FANCI-FANCD2 substrate. Crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1. A cryoEM reconstruction of USP1-UAF1 in complex mono-ubiquitinated FANCI-FANCD2, highlights a highly orchestrated deubiquitination process with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for their requirement despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

A Potential Novel Therapeutic Approach for Fanconi Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1040-1040
Author(s):  
W. Clark Lambert ◽  
Monique M Brown
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Normal Subjects ◽  
High Dose ◽  
Inherited Disease ◽  
Trypan Blue Exclusion ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks ◽  
Cellular Dna

Abstract Lymphoblastoid cells from normal subjects and from patients with the bone marrow failure and cancer prone inherited disease, Fanconi anemia (FA) were treated in culture with psoralen plus ultraviolet A radiation (PUVA) in a scheme shown to produce interstrand crosslinks in cellular DNA. Hypersensitivity to DNA interstrand crosslinks, with associated increased clastogenicity, is considered to be a diagnostic hallmark of the disease. Following this cells were treated with hydroxyurea, 5 fluorouracil, or high dose thymidine for 24 hours. Clastogenicity and cytotoxicity, measured as trypan blue exclusion, were then found to be markedly increased in FA cells but not in FA cells subsequently treated with any of these other agents. Similar results were also found when all drugs were removed after these treatments and the cells cultured for 10 days without any drug in colony forming ability assays. We propose that the mechanism is related to decrease in the rate of DNA synthesis, which we have shown occurs in normal but not FA cells following PUVA, and which is also produced by these other drugs in the concentrations used here. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis, with or without prior bone marrow transplantation.

Sign in / Sign up

Export Citation Format

Share Document