scholarly journals Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Winnie Tan ◽  
Sylvie van Twest ◽  
Andrew Leis ◽  
Rohan Bythell-Douglas ◽  
Vincent J Murphy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Critical Event ◽  
Core Complex ◽  
Binding Motifs ◽  
Ubiquitin Binding ◽  
Functional Consequences ◽  
Cancer Types ◽  
Stalled Replication Forks

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.

scholarly journals Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

2019 ◽  
Author(s):  
Winnie Tan ◽  
Sylvie van Twest ◽  
Andrew Leis ◽  
Rohan Bythell-Douglas ◽  
Vincent J. Murphy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Critical Event ◽  
Replication Forks ◽  
Core Complex ◽  
Binding Motifs ◽  
Ubiquitin Binding ◽  
Functional Consequences ◽  
Stalled Replication Forks

AbstractFANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This locking of FANCI:FANCD2 complex on DNA requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2 is activated upon DNA binding and present new insights to potentially modulate monoubiquitinated FANCI:FANCD2/DNA filaments in FA cells.

scholarly journals FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3285-3294 ◽  
Author(s):  
Abdullah Mahmood Ali ◽  
Arun Pradhan ◽  
Thiyam Ramsingh Singh ◽  
Changhu Du ◽  
Jie Li ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Core Complex ◽  
Functional Integrity ◽  
Isolation And Characterization ◽  
Nuclear Core ◽  
Damage Repair ◽  
Ubiquitin Binding ◽  
Complex Protein

Abstract Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63–linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage–induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.

Selected Resistance to Topoisomerase II Inhibitors Correlates with the Over Expression of FANCF In a Fanconi Anemia/BRCA DNA Repair Pathway Independent Manner.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3372-3372
Author(s):  
Kenneth H. Shain ◽  
Liang Nong ◽  
Danielle Yarde ◽  
Vasco Oliveira ◽  
William S. Dalton
Keyword(s):  
Dna Repair ◽  
Mrna Expression ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Repair Pathway ◽  
Core Complex ◽  
Doxorubicin Resistance ◽  
Over Expression ◽  
The Core ◽  
Enhanced Expression

Abstract Abstract 3372 Enhanced expression of the Fanconi Anemia (FA)/BRCA DNA repair pathway correlates with melphalan-resistance in multiple myeloma (MM) cell lines. Continued investigation demonstrated a bortezomib sensitive RelB/p50-mediated regulation of the FA/BRCA pathway contributed to the observed melphalan resistance.(Yarde et al 2009) The FA/BRCA pathway represents a co-dependent DNA damage response pathway involving thirteen loss of function complementation groups cloned from FA patients. The key functional event of this pathway is the interdependent mono-ubiquitination (Ub) of FANCD2 and FANCI (ID complex) by the E3 Ub-ligase activity of the FA core complex a multimer consisting of 8 FA (FANCA, B, C, E, F, G, L and M) and three non-FA proteins (FAAP100, FAAP24 and HES1). Formation of the core complex and mono-Ub of the ID complex appears to revolve around the flexible adapter protein FANCF. Nuclear localization of the core complex components requires binding of FANCA/G and FANCC/E subcomplexes to the C- terminal domain (CTD) and NTD domains of FANCF, respectively. This complex associates with FANCM:FAAP24 at sites of interstand crosslinks (ICL) via the FANCM-binding domain of FANCF, culminating in ID complex mono-Ub, recruitment of BRCA1, BRCA2/FANCD1, FANCJ and FANCN, and homologous recombination (HR) repair. Reduced function of this pathway has been associated with increased genomic instability, cancer susceptibility, and increased sensitivity to DNA cross-linking agents in FA. However, as predicted by the role of the FA/BRCA pathway in DNA repair, enhanced expression of the FA/BRCA pathway has been shown to play an important role in resistance to agents requiring HR for ICL repair. We next examined expression of this pathway in models of resistance to DNA damaging agents not predicted to utilize FA/BRCA activity. We screened 8226/Dox40 doxorubicin resistant and 8226/MR20 mitoxantrone resistant MM cell lines for expression of the 12 FA/BRCA pathway members with quantitative PCR (qPCR) using customized micro-fluidic cards. Interestingly, in these models of topoisomerase (topo) II inhibitor resistance qPCR demonstrated a 2.6 (p<0.05) and 1.7 (p<0.05) fold over expression of FANCF mRNA relative to drug sensitive RPMI8226 cells. Importantly, mRNA expression of other the eleven FA/BRCA pathway constituents was not increased relative to sensitive cells. To further characterize the relationship between FANCF and doxorubicin resistance, we examined mRNA and protein expression of FANCF in RMPI8226, 8226/Dox6 and 8226/Dox40 MM cell lines (representing progressive levels of doxorubicin resistance). FANCF qPCR demonstrated a 2 and 4.7 fold increased in mRNA expression in the 8226/Dox6 and 8226/Dox40 cell lines, respectively (p= 0.103 and p= 0.034) suggesting that increasing expression of FANCF correlated with increasing dox resistance. A similar doxorubicin resistance- dependent increase in FANCF protein was demonstrated by Western blot analysis of these cell lines. Consistent with mRNA results, FANCD2 or FANCG protein levels remained unchanged in the doxorubicin resistant versus sensitive cell lines These observations suggest that FANCF may contribute to topoII inhibitor-mediated DNA double strand break repair, a process that primarily thought to involve non-homologous end joining (NHEJ) independent of the FA/BRCA pathway. To determine if FANCF expression alone could facilitate doxorubicin resistance, pQCXIP-control or pQCXIP-FANCF constructs were expressed in RPMI8226 sensitive MM cells. MTT assays demonstrated a greater than 2 fold resistance to doxorubicin in FANCF over expressing cells at 48 and 96 hours (IC50: 1.33 ×10−6 and 5.3×10−9M) as compared to control cells (3.26×10−6 and 1.13×10−8M). Taken together, these results indicate that the flexible adaptor protein FANCF may participate in doxorubicin resistance independently of other FA/BRCA members. However, future studies will be needed to elucidate the nature of FANCF in doxorubicin resistance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2109-2117 ◽  
Author(s):  
Meghan A. Rego ◽  
Frederick W. Kolling ◽  
Elizabeth A. Vuono ◽  
Maurizio Mauro ◽  
Niall G. Howlett
Keyword(s):  
Fanconi Anemia ◽  
Noncovalent Interaction ◽  
Binding Domain ◽  
Covalent Attachment ◽  
Pathway Activation ◽  
Ubiquitin Binding ◽  
Ubiquitin Binding Domain ◽  
Ubiquitin Conjugation ◽  
Cue Domain

Abstract The Fanconi anemia (FA)–BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that although mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2, and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation.

scholarly journals FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair

2008 ◽  
Vol 17 (11) ◽  
pp. 1641-1652 ◽  
Author(s):  
Yutong Xue ◽  
Yongjiang Li ◽  
Rong Guo ◽  
Chen Ling ◽  
Weidong Wang
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Core Complex

scholarly journals A FancD2-Monoubiquitin Fusion Reveals Hidden Functions of Fanconi Anemia Core Complex in DNA Repair

2005 ◽  
Vol 19 (6) ◽  
pp. 841-847 ◽  
Author(s):  
Nobuko Matsushita ◽  
Hiroyuki Kitao ◽  
Masamichi Ishiai ◽  
Naoki Nagashima ◽  
Seiki Hirano ◽  
...  
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Core Complex

scholarly journals Targeting the Fanconi Anemia Pathway to Identify Tailored Anticancer Therapeutics

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Chelsea Jenkins ◽  
Jenny Kan ◽  
Maureen E. Hoatlin
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Fanconi Anemia ◽  
Cancer Therapeutics ◽  
Core Complex ◽  
Synthetic Lethal ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Repair Pathways ◽  
Repair Genes

The Fanconi Anemia (FA) pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs). The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.

scholarly journals Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1116-1116
Author(s):  
Abeer Najjar
Keyword(s):  
Dna Repair ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
Hela Cells ◽  
Repair Pathway ◽  
Core Complex ◽  
Target Proteins ◽  
Ubiquitinated Proteins ◽  
Ubiquitin Ligase Complex

Abstract The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. A pivotal step in the activation of the FA DNA repair pathway is the monoubiquitination of the FANCD2 and FANCI proteins (ID2) by the FA core complex, a unique ubiquitin ligase complex which includes eight proteins (FANCA-FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. Cells with mutations in any of the FA core complex proteins lack the ability to monoubiquitinated ID2, making ID2 ubiquitination a convergence point in the pathway, with an estimation of&gt;90% FA patients defective in this step. Additionally, somatic mutations In FA genes render tumor cells sensitive to DNA crosslinking agents, so identification of FA pathway defects provides an opportunity for therapeutic targeting. In search for additional potential target/substrate of this unique FA core ubiquitin ligase complex, we performed a high throughput genome-wide ubiquitin-specific proteomics (UbiScan) screen and found, in addition to the ID2 complex, many ubiquitinated proteins are dysregulated (mostly downregulated) in FA deficient cells compared with that of FA proficient cells. We used a Ubiquitin Remnant Motif (K- ∑-GG) Antibody Bead Conjugate (Cell Signaling Technology), a proprietary ubiquitin branch ("K- ∑-GG") antibody with specificity for a di-glycine tag that is the remnant of ubiquitin left on protein substrates after trypsin digestion, to enrich ubiquitinated peptides from trypsin digested cell samples (shNT vs shFANCA). This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand nonredundant ubiquitinated sequences. We were successful in demonstrating that under steady-state conditions (without proteasome inhibitor treatment), the ubiquitinated forms of both FANCD2 and FANCI proteins are much higher in control (shNT) HeLa cells compared with that of the cells depleted of FANCA (shFANCA). We then collaborated with the Cell signaling technology to perform a high throughput UbiScan® analysis of total ubiquitinated proteins both in total nuclei and chromatin fractions under replicative stress conditions. UbiScan® enables researchers to isolate, identify and quantitate large numbers of ubiquitin-modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of the ubiquitination sites in cellular proteins in cell and tissue samples without preconceived biases about where these modified sites occur. A total of 16,249 redundant modified peptide assignments to 7,856 modified sites for the Ubiquitin K-GG Remnant Motif Antibody were obtained. As expected, the amount of monoubiquitinated FANCD2 (at K651) and FANCI (at K523) were highly reduced in both the nuclear and chromatin fractions of Hela cells depleted of FANCA (shA). Consistent with the earlier findings, the amount of ubiquitinated ID2 proteins were extremely low in the chromatin fraction of the Hela cells depleted of FANCA. Since there are numerous ubiquitinated proteins found to be dysregulated in our UbiScan analyses, we used the following criteria to select the target proteins based on; a) -fold changes, and b) proteins that are known to participate in the DNA repair signaling pathways. We validated our UbiScan results by using an assay system to detect endogenous protein ubiquitination. We also found a significant reduction in the ubiquitination of several DNA repair-related proteins (found in our UbiScan analysis) in FANCA deficient cells. To assess FA pathway functions, we generated HAP1 and appropriate cells knock out of these select ubiquitinated target proteins by using CRISPR-Cas9 system. Then, the KO cells were examined for FA pathway functions. These results will be discussed. In conclusion, our findings reveal that the FA core ubiquitin ligase complex regulates (directly or indirectly) the ubiquitinated levels of many novel proteins outside of the ID2 complex, and these novel target proteins may provide important additional mechanistic insights into the FA DNA repair pathway. Disclosures No relevant conflicts of interest to declare.

scholarly journals New Phosphorylation Sites of Rad51 by c-Met Modulates Presynaptic Filament Stability

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 413 ◽  
Author(s):  
Thomas Chabot ◽  
Alain Defontaine ◽  
Damien Marquis ◽  
Axelle Renodon-Corniere ◽  
Emmanuelle Courtois ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Tyrosine Kinases ◽  
Post Translational Modifications ◽  
Subunit Interface ◽  
Central Protein ◽  
Cancer Types ◽  
First Time

Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response.

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