DOCSLIB.ORG

Fanconi Anemia (FA) Binding Protein FAAP20 Stabilizes FA Complementation Group a (FANCA) and Participates in Interstrand Cross-Link Repair

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Fanconi Anemia (FA) Binding Protein FAAP20 Stabilizes FA Complementation Group a (FANCA) and Participates in Interstrand Cross-Link Repair Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair Justin Wai Chung Leunga, Yucai Wanga, Ka Wing Fonga, Michael Shing Yan Huenb, Lei Lia, and Junjie Chena,1 aDepartment of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030; and bGenome Stability Research Laboratory, Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region, China Edited* by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved February 6, 2012 (received for review November 13, 2011) The Fanconi anemia (FA) pathway participates in interstrand cross- proliferating cell nuclear antigen (PCNA) monoubiquitination link (ICL) repair and the maintenance of genomic stability. The FA (26). It remains to be resolved how PCNA monoubiquitination is core complex consists of eight FA proteins and two Fanconi anemia- linked with the activation of the FA pathway. FANCA ∼ associated proteins (FAAP24 and FAAP100). The FA core complex Among all of the FA genes, mutations in ( 60%), FANCC (∼14%), and FANCG (∼10%) account for over 80% of has ubiquitin ligase activity responsible for monoubiquitination of fi the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of the mutations identi ed in patients (27). However, FANCA, biochemical events that allow processing and removal of cross- FANCC, and FANCG are orphan proteins that do not share extensive sequence homology with other proteins. Thus, it is still linked DNA and thereby promotes cell survival following DNA fi unknown how these proteins function in the FA pathway. We damage. Here, we report the identi cation of a unique component reason that the identification of new FA-associated proteins may of the FA core complex, namely, FAAP20, which contains a RAD18- fi help us understand how these orphan proteins participate in like ubiquitin-binding zinc- nger domain. Our data suggest that DNA repair. In this study, we report the identification of C1orf86 FAAP20 promotes the functional integrity of the FA core complex isoform2 as a previously undescribed FANCA-interacting protein via its direct interaction with the FA gene product, FANCA. Indeed, (Fanconi anemia-associated protein 20 kDa, hereafter referred as somatic knockout cells devoid of FAAP20 displayed the hallmarks FAAP20). Genetic inactivation of FAAP20 revealed many fea- of FA cells, including hypersensitivity to DNA cross-linking agents, tures of FA cells, highlighting that FAAP20 is a key component of chromosome aberrations, and reduced FANCD2 monoubiquitina- the FA core complex and participates in ICL repair. tion. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway. Results FAAP20 Is a Unique Component of the FA Core Complex. We per- mitomycin C | DNA repair | foci formed tandem affinity purification (TAP) using FANCA as bait to identify FANCA-associated proteins. After excluding general con- taminants, such as heat-shock proteins and ribosomal proteins, we anconi anemia (FA) is a rare recessive genetic disorder char- fi A Facterized by bone marrow failure, congenital developmental identi ed FAAP20 as a potential FANCA-binding partner (Fig. 1 ). defects, and cancer predisposition (1–4). Cellular features of FA FAAP20 (LOC1999990 isoform 2) encodes a 20-kDa protein with unknown function (gene name: C1orf86 isoform 2; accession number: include chromosomal instability and hypersensitivity to cross- fi linking agents (5). Fifteen FA complementation genes have been NP_872339.2). To con rm its association with FANCA, we per- fi formed a reverse purification using FAAP20 as bait and showed that identi ed so far. These genes form several complexes to orches- fi A trate interstrand cross-linking (ICL) repair. The FA core complex FANCA and FANCG copuri ed with FAAP20 (Fig. 1 ), indicating is composed of eight of the FA gene products (FANCA, FANCB, that FAAP20 is a potential component of the FA core complex. FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM), in addition to FAAP24 and FAAP100 (6, 7), and acts as an E3 ligase FAAP20 Binds Directly to FANCA. To verify that FAAP20 interacts to ubiquitinate FANCI/FANCD2 (I/D2) complex (8–11). The with FANCA, we coexpressed triple-tagged (SFB-tag: S-protein tag, monoubiquitinated FANCI/FANCD2 complex interacts with FLAG epitope, tag and streptavidin-binding peptide tag) FANCA Fanconi anemia-associated nuclease 1 (FAN1), which has exo- with Myc-tagged FAAP20 or FANCG. As expected, we observed nuclease and endonuclease activity that may unhook the ICL, a robust binding of SFB-tagged FANCA with Myc-tagged FANCG. In addition, we found a strong interaction between FANCA and facilitate translesion synthesis, and promote downstream homol- B ogous recombination (HR) repair (12–15). FAAP20 (Fig. 1 ), suggesting that they exist in the same complex. Besides the FA core complex and FANCI/FANCD2, there are Moreover, we showed that endogenous FANCA coimmunopreci- pitated with endogenous FAAP20 and this interaction occurs in- several other FA proteins that likely act downstream of FANCI/ C FANCD2 and participate in HR repair. These proteins include dependently of mitomycin C (MMC) treatment (Fig. 1 ). We generated a series of internal deletion mutants of FANCA BRCA2 (FANCD1) and PALB2 (FANCN), both of which are D essential for HR repair (16–18). Another downstream FA pro- (FANCA-D1 to FANCA-D5) (Fig. 1 ) and observed that two of fi them, FANCA-D3 and FANCA-D4, failed to interact with tein, BACH1 (FANCJ), is also a bona de double-strand break E repair factor (19). BRCA2/FANCD1, PALB2/FANCN, and FAAP20 (Fig. 1 ), indicating that FAPP20 binds to the middle BACH1/FANCJ are all recruited to ICL sites (20), indicating that region of FANCA. In addition, we performed a pull-down assay they are directly involved in ICL repair. More recently, mutations using bacterial expressed GST-fused FAAP20 and maltose- in two other genes, RAD51C/FANCO and SLX4/FANCP, were CELL BIOLOGY identified in patients with FA phenotypes (21–23), suggesting that there may be additional FA genes responsible for this disease. Author contributions: J.W.C.L., M.S.Y.H., L.L., and J.C. designed research; J.W.C.L., Y.W., Not all FA proteins function in a linear pathway involved in ICL M.S.Y.H., and K.W.F. performed research; J.W.C.L., Y.W., K.W.F., M.S.Y.H., L.L., and J.C. repair. Many of the downstream FA proteins are involved in HR analyzed data; and J.W.C.L., Y.W., K.W.F., M.S.Y.H., L.L., and J.C. wrote the paper. repair and associated with breast cancer susceptibility (16–19, 24, The authors declare no conflict of interest. 25). These proteins all have functions besides ICL repair. More *This Direct Submission article had a prearranged editor. recently, another DNA repair protein, RAD18, which is best known 1To whom correspondence should be addressed. E-mail: [email protected]. for its role in UV lesion bypass, has also been shown to participate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the activation of the FA pathway via its ability to promote 1073/pnas.1118720109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118720109 PNAS | March 20, 2012 | vol. 109 | no. 12 | 4491–4496 Downloaded by guest on October 2, 2021 AB SFB-FANCA Myc-FANCG Myc-FAAP20 WB: Flag (FANCA) FANCG WB: Myc FAAP20 Fig. 1. FAAP20 is a FANCA-binding protein. (A) 293T cells 2% Input Pull down: stably expressed SFB-FANCA or SFB-FAAP20, respectively, Streptividine were subjected to TAP and mass spectrometry analysis. Red beads indicates the bait protein and blue indicates the known or putative-associated proteins. Number of peptides recovered from mass spectrometry analysis is also presented. (B) 293T C AAP20 cells were transfected with constructs encoding SFB-FANCA FANCA 2% Input IP:IgG IP:F along with constructs encoding Myc-FANCG or Myc-FAAP20. D FANCA MMC FANCA 11-673) WB: FANCA Coprecipitation and immunoblotting were carried out as FANCA indicated. (C) Lysates prepared from control or MMC-treated FANCA WB: FAAP20 293T cells were incubated with protein A agarose beads FANCA conjugated with rabbit IgG or anti-FAAP20 antibodies. P 2 0 Western blotting was performed using indicated antibodies. Pulldown EFI n p u t 10% Input Streptavidine beads A (D) Schematic illustration of wild-type and deletion mutants -FA - D 1 A N C A - D 2 3 D 4 A N C A - D 5 F A N C A - D 1 2 C A - D 3 - D 4 D 5 of FANCA used in this study. (E) 293T cells were transfected % T GST FANCA-D CA-D FANCA- - GS NCA CA FANCA FANCA A with constructs encoding SFB-FAAP20 along with constructs 2 FANC FAN FAN AN WB: MBP (FANCG) encoding Myc-tagged wild-type or deletion mutants of A F F F F WB: MBP FAAP20. Precipitation and immunoblotting were conducted WB:Flag (FANCA 674-1208) (FAAP20) as indicated. (F) Pull-down assays were performed using bacterially expressed and purified GST-FAAP20 and MBP- WB:Myc Coomassie (FANCA) Staining fused FANCA (residues 674–1032) or FANCG. Immunoblot- ting were conducted using anti-MBP antibody. binding protein (MBP)-fused FANCA (aa674-1208) or MBP-fused FANCA (Fig. 2D), suggesting that this N-terminal motif within FANCG. GST-FAAP20 pulled down MBP-fused FANCA (resi- FAAP20 is necessary for FAAP20-FANCA interaction. dues 674–1208), but not MBP-fused FANCG (Fig. 1F), suggesting that FAAP20 binds directly to FANCA, but not to FANCG. FAAP20 Contains an Evolutionarily Conserved RAD18-like Ubiquitin- We then used a series of FAAP20 internal-deletion mutants to Binding Zinc-Finger Domain. We noticed that FAAP20 contains an map a FANCA-binding region on FAAP20 (Fig.
Load more

Recommended publications
  • Structure and Function of the Human Recq DNA Helicases
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2005 Structure and function of the human RecQ DNA helicases Garcia, P L Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-34420 Dissertation Published Version Originally published at: Garcia, P L. Structure and function of the human RecQ DNA helicases. 2005, University of Zurich, Faculty of Science. Structure and Function of the Human RecQ DNA Helicases Dissertation zur Erlangung der naturwissenschaftlichen Doktorw¨urde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultat¨ der Universitat¨ Z ¨urich von Patrick L. Garcia aus Unterseen BE Promotionskomitee Prof. Dr. Josef Jiricny (Vorsitz) Prof. Dr. Ulrich H ¨ubscher Dr. Pavel Janscak (Leitung der Dissertation) Z ¨urich, 2005 For my parents ii Summary The RecQ DNA helicases are highly conserved from bacteria to man and are required for the maintenance of genomic stability. All unicellular organisms contain a single RecQ helicase, whereas the number of RecQ homologues in higher organisms can vary. Mu- tations in the genes encoding three of the five human members of the RecQ family give rise to autosomal recessive disorders called Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome. These diseases manifest commonly with genomic in- stability and a high predisposition to cancer. However, the genetic alterations vary as well as the types of tumours in these syndromes. Furthermore, distinct clinical features are observed, like short stature and immunodeficiency in Bloom syndrome patients or premature ageing in Werner Syndrome patients. Also, the biochemical features of the human RecQ-like DNA helicases are diverse, pointing to different roles in the mainte- nance of genomic stability.
    [Show full text]
  • What Is Fanconi Anemia and How Is It Diagnosed
    Fanconi anemia and its diagnosis: Fanconi anemia (FA), named for the Swiss pediatrician Guido Fanconi, is an inherited disorder that can lead to bone marrow failure (aplastic anemia), leukemia and/or solid tumors, with oral and gynecologic tumors being the most common. FA is almost exclusively a recessive disorder: if both parents carry a defect (mutation) in the same FA gene, each of their children has a 25% chance of inheriting the defective gene from both parents. When this happens, the child will have FA. While the total number of FA patients is not documented worldwide, scientists estimate that the carrier frequency (carriers are people carrying a defect in one copy of a particular FA gene, whose other copy of that same FA gene is normal) for FA in the U.S. is 1 in 181. The incidence rate, or the likelihood of a child being born with FA, is about 1 in 131,000 in the U.S., with approximately 31 babies born with FA each year. Scientists have now discovered 19 FA genes [FANCA, -B, -C, -D1 (also known as BRCA2), D2, E, F, G, I, J, L, M, N, O, P, Q, RAD51, BRCA1, and T]. Mutations in these genes account for more than 95% of reported Fanconi anemia cases. Mutations in FANCA, FANCC and FANCG are the most common and account for approximately 85% of FA patients worldwide. FANCD1, FANCD2, FANCE, FANCF and FANCL account for 10%, while the remaining FA genes, FANCB, FANCI, FANCJ, FANCM, FANCN, FANCO, FANCP, and FANCQ represent less than 5%. Some individuals with FA do not appear to have mutations in these 19 genes, so we anticipate that additional FA genes will be discovered in the future.
    [Show full text]
  • A Novel Breast Cancer ^ Associated BRIP1 (FANCJ/BACH1) Germ- Line Mutation Impairs Protein Stability and Function
    Cancer Prevention and Susceptibility A Novel Breast Cancer ^ Associated BRIP1 (FANCJ/BACH1)Germ- line Mutation Impairs Protein Stability and Function Arcangela De Nicolo,1MariellaTancredi,4 Grazia Lombardi,4 Cristina Chantal Flemma,4 Serena Barbuti,4 Claudio Di Cristofano,4 Bijan Sobhian,1Generoso Bevilacqua,4 Ronny Drapkin,2,3 andMariaAdelaideCaligo4 Abstract Purpose: BRCA1-interacting protein 1 (BRIP1; FANCJ/BACH1), which encodes a DNA helicase that interacts with BRCA1, has been suggested to be a low-penetrance breast cancer predispos- ing gene.We aimed to assess whether BRIP1 mutations contribute to breast cancer susceptibility in our population and, if so, to investigate the effect of such mutation(s) on BRIP1function. Experimental Design: A series of49 breast/ovarian cancer families, devoid ofa BRCA1/ BRCA2 mutation, were screened for BRIP1 mutations. Functional analyses, including coimmuno- precipitation and stability assays, were employed to further characterize a previously unreported variant. Results: Five sequence alterations were identified, of which four had been already described. Herein, we report a novel BRIP1 germ-line mutation identified in a woman with early-onset breast cancer. The mutation consists ofa 4-nucleotide deletion (c.2992-2995delAAGA) in BRIP1 exon 20 that causes a shift in the reading frame, disrupts the BRCA1-binding domain of BRIP1, and creates a premature stop codon. Functional analysis ofthe recombinant mutant protein in transfected cells showed that the truncation interferes with the stability of the protein and with its ability to interact with BRCA1. Loss ofthe wild-type BRIP1 allele with retention ofthe mutated one was observed in the patient’s breast tumor tissue. Conclusions: These results, by showing that the newly identified BRIP1 c.2992-2995delAAGA mutation is associated with instability and functional impairment of the encoded protein, provide further evidence of a breast cancer ^ related role for BRIP1.
    [Show full text]
  • Open Full Page
    CCR PEDIATRIC ONCOLOGY SERIES CCR Pediatric Oncology Series Recommendations for Childhood Cancer Screening and Surveillance in DNA Repair Disorders Michael F. Walsh1, Vivian Y. Chang2, Wendy K. Kohlmann3, Hamish S. Scott4, Christopher Cunniff5, Franck Bourdeaut6, Jan J. Molenaar7, Christopher C. Porter8, John T. Sandlund9, Sharon E. Plon10, Lisa L. Wang10, and Sharon A. Savage11 Abstract DNA repair syndromes are heterogeneous disorders caused by around the world to discuss and develop cancer surveillance pathogenic variants in genes encoding proteins key in DNA guidelines for children with cancer-prone disorders. Herein, replication and/or the cellular response to DNA damage. The we focus on the more common of the rare DNA repair dis- majority of these syndromes are inherited in an autosomal- orders: ataxia telangiectasia, Bloom syndrome, Fanconi ane- recessive manner, but autosomal-dominant and X-linked reces- mia, dyskeratosis congenita, Nijmegen breakage syndrome, sive disorders also exist. The clinical features of patients with DNA Rothmund–Thomson syndrome, and Xeroderma pigmento- repair syndromes are highly varied and dependent on the under- sum. Dedicated syndrome registries and a combination of lying genetic cause. Notably, all patients have elevated risks of basic science and clinical research have led to important in- syndrome-associated cancers, and many of these cancers present sights into the underlying biology of these disorders. Given the in childhood. Although it is clear that the risk of cancer is rarity of these disorders, it is recommended that centralized increased, there are limited data defining the true incidence of centers of excellence be involved directly or through consulta- cancer and almost no evidence-based approaches to cancer tion in caring for patients with heritable DNA repair syn- surveillance in patients with DNA repair disorders.
    [Show full text]
  • Analysis of and Fanconi Anemia Genes in -Negative Spanish Breast Cancer Families María J
    Analysis of and Fanconi Anemia genes in -negative Spanish breast cancer families María J. García, Victoria Fernández, Ana Osorio, Alicia Barroso, Gemma Llort, Conxi Lázaro, Ignacio Blanco, Trinidad Caldés, Miguel Hoya, Teresa Ramón y Cajal, et al. To cite this version: María J. García, Victoria Fernández, Ana Osorio, Alicia Barroso, Gemma Llort, et al.. Analysis of and Fanconi Anemia genes in -negative Spanish breast cancer families. Breast Cancer Research and Treatment, Springer Verlag, 2008, 113 (3), pp.545-551. 10.1007/s10549-008-9945-0. hal-00478320 HAL Id: hal-00478320 https://hal.archives-ouvertes.fr/hal-00478320 Submitted on 30 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Breast Cancer Res Treat (2009) 113:545–551 DOI 10.1007/s10549-008-9945-0 EPIDEMIOLOGY Analysis of FANCB and FANCN/PALB2 Fanconi Anemia genes in BRCA1/2-negative Spanish breast cancer families Marı´a J. Garcı´a Æ Victoria Ferna´ndez Æ Ana Osorio Æ Alicia Barroso Æ Gemma LLort Æ Conxi La´zaro Æ Ignacio Blanco Æ Trinidad Calde´s Æ Miguel de la Hoya Æ Teresa Ramo´n y Cajal Æ Carmen Alonso Æ Marı´a-Isabel Tejada Æ Carlos San Roma´n Æ Luis Robles-Dı´az Æ Miguel Urioste Æ Javier Benı´tez Received: 12 February 2008 / Accepted: 12 February 2008 / Published online: 27 February 2008 Ó Springer Science+Business Media, LLC.
    [Show full text]
  • The ATM Gene in Breast Cancer: Its Relevance in Clinical Practice
    G C A T T A C G G C A T genes Review The ATM Gene in Breast Cancer: Its Relevance in Clinical Practice Luigia Stefania Stucci 1,* , Valeria Internò 1 , Marco Tucci 1,2 , Martina Perrone 1, Francesco Mannavola 1 , Raffaele Palmirotta 3 and Camillo Porta 1 1 Division of Medical Oncology, Department of Biomedical Sciences and Human Oncology, University of Bari ‘Aldo Moro’, A.O.U. Consorziale Policlinico di Bari, 70121 Bari, Italy; [email protected] (V.I.); [email protected] (M.T.); [email protected] (M.P.); [email protected] (F.M.); [email protected] (C.P.) 2 National Cancer Research Center, Tumori Institute IRCCS Giovanni Paolo II, 70121 Bari, Italy 3 Interdisciplinary Department of Medicine, Section of Sciences and Technologies of Laboratory Medicine, University of Bari, 70121 Bari, Italy; [email protected] * Correspondence: [email protected] Abstract: Molecular alterations of the Ataxia-telangiectasia (AT) gene are frequently detected in breast cancer (BC), with an incidence ranging up to 40%. The mutated form, the Ataxia-telangiectasia mutated (ATM) gene, is involved in cell cycle control, apoptosis, oxidative stress, and telomere maintenance, and its role as a risk factor for cancer development is well established. Recent studies have confirmed that some variants of ATM are associated with an increased risk of BC development and a worse prognosis. Thus, many patients harboring ATM mutations develop intermediate- and high-grade disease, and there is a higher rate of lymph node metastatic involvement. The evidence concerning a correlation of ATM gene mutations and the efficacy of therapeutic strategies in BC management are controversial.
    [Show full text]
  • About PALB2 Gene Mutations
    About PALB2 Gene Mutations About Genes Recommendations Genes are in every cell in our bodies. Genes are made WOMEN of DNA, which gives instructions to cells about how to Starting at age 30: Mammogram and breast MRI every grow and work together. We have two copies of each year (scheduled 6 months apart) gene in each cell—one from our mother and one from our father. When genes work properly, they help stop Some medicines can lower the risk of getting breast cancer from developing. cancer. Surgery to remove both breasts may be an option for some women who have a strong family history of When it works right, the PALB2 gene works together breast cancer. with the BRCA1 and BRCA2 genes to help prevent WOMEN AND MEN cancer. Sometimes changes to the PALB2 gene happen. These changes are called mutations. Mutations can make Screening for pancreatic cancer has benefits and the PALB2 gene stop working and raise the risk for limitations. We do not recommend this screening for certain types of cancer. most people with PALB2 mutations. People who have a PALB2 mutation and a family history of pancreatic Having a mutation in the PALB2 gene makes your risk cancer should ask their doctor or genetic counselor for of getting breast and pancreatic cancers higher than more information. average. The risks for other cancers may also go up with KIDS AND SIBLINGS PALB2 mutations. Researchers are studying the PALB2 gene to understand more. Children and siblings of people with a PALB2 mutation have a 1 in 2 chance of also having the mutation.
    [Show full text]
  • FANCL Sirna (H): Sc-45661
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . FANCL siRNA (h): sc-45661 BACKGROUND STORAGE AND RESUSPENSION Defects in FANCL are a cause of Fanconi anemia. Fanconi anemia (FA) is an Store lyophilized siRNA duplex at -20° C with desiccant. Stable for at least autosomal recessive disorder characterized by bone marrow failure, birth one year from the date of shipment. Once resuspended, store at -20° C, defects and chromosomal instability. At the cellular level, FA is characterized avoid contact with RNAses and repeated freeze thaw cycles. by spontaneous chromosomal breakage and a unique hypersensitivity to DNA Resuspend lyophilized siRNA duplex in 330 µl of the RNAse-free water cross-linking agents. At least 8 complementation groups have been identified pro vided. Resuspension of the siRNA duplex in 330 µl of RNAse-free water and 6 FA genes (for subtypes A, C, D2, E, F and G) have been cloned. Phospho- makes a 10 µM solution in a 10 µM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM rylation of FANC (Fanconi anemia complementation group) proteins is thought EDTA buffered solution. to be important for the function of the FA pathway. FA proteins cooperate in a common pathway, culminating in the monoubiquitination of FANCD2 protein APPLICATIONS and colocalization of FANCD2 and BRCA1 proteins in nuclear foci. FANCL is a ligase protein mediating the ubiquitination of FANCD2, a key step in the FANCL shRNA (h) Lentiviral Particles is recommended for the inhibition of DNA damage pathway. FANCL may be required for proper primordial germ cell FANCL expression in human cells.
    [Show full text]
  • Genomic Profiling Reveals High Frequency of DNA Repair Genetic
    www.nature.com/scientificreports OPEN Genomic profling reveals high frequency of DNA repair genetic aberrations in gallbladder cancer Reham Abdel‑Wahab1,9, Timothy A. Yap2, Russell Madison4, Shubham Pant1,2, Matthew Cooke4, Kai Wang4,5,7, Haitao Zhao8, Tanios Bekaii‑Saab6, Elif Karatas1, Lawrence N. Kwong3, Funda Meric‑Bernstam2, Mitesh Borad6 & Milind Javle1,10* DNA repair gene aberrations (GAs) occur in several cancers, may be prognostic and are actionable. We investigated the frequency of DNA repair GAs in gallbladder cancer (GBC), association with tumor mutational burden (TMB), microsatellite instability (MSI), programmed cell death protein 1 (PD‑1), and its ligand (PD‑L1) expression. Comprehensive genomic profling (CGP) of 760 GBC was performed. We investigated GAs in 19 DNA repair genes including direct DNA repair genes (ATM, ATR , BRCA1, BRCA2, FANCA, FANCD2, MLH1, MSH2, MSH6, PALB2, POLD1, POLE, PRKDC, and RAD50) and caretaker genes (BAP1, CDK12, MLL3, TP53, and BLM) and classifed patients into 3 groups based on TMB level: low (< 5.5 mutations/Mb), intermediate (5.5–19.5 mutations/Mb), and high (≥ 19.5 mutations/Mb). We assessed MSI status and PD‑1 & PD‑L1 expression. 658 (86.6%) had at least 1 actionable GA. Direct DNA repair gene GAs were identifed in 109 patients (14.2%), while 476 (62.6%) had GAs in caretaker genes. Both direct and caretaker DNA repair GAs were signifcantly associated with high TMB (P = 0.0005 and 0.0001, respectively). Tumor PD‑L1 expression was positive in 119 (15.6%), with 17 (2.2%) being moderate or high. DNA repair GAs are relatively frequent in GBC and associated with coexisting actionable mutations and a high TMB.
    [Show full text]
  • Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein
    University of Rhode Island DigitalCommons@URI Open Access Dissertations 2015 Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein Rebecca Anne Boisvert University of Rhode Island, [email protected] Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss Recommended Citation Boisvert, Rebecca Anne, "Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein" (2015). Open Access Dissertations. Paper 397. https://digitalcommons.uri.edu/oa_diss/397 This Dissertation is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. FURTHER INSIGHTS INTO THE REGULATION OF THE FANCONI ANEMIA FANCD2 PROTEIN BY REBECCA ANNE BOISVERT A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL AND MOLECULAR BIOLOGY UNIVERSITY OF RHODE ISLAND 2015 DOCTOR OF PHILOSOPHY DISSERTATION OF REBECCA ANNE BOISVERT APPROVED: Dissertation Committee: Major Professor Niall Howlett Paul Cohen Becky Sartini Nasser H. Zawia DEAN OF THE GRADUATE SCHOOL UNIVERSITY OF RHODE ISLAND 2015 ABSTRACT Fanconi anemia (FA) is a rare autosomal and X-linked recessive disorder, characterized by congenital abnormalities, pediatric bone marrow failure and cancer susceptibility. FA is caused by biallelic mutations in any one of 16 genes. The FA proteins function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). The monoubiquitination of FANCD2 and FANCI is a central step in the activation of the FA-BRCA pathway and is required for targeting these proteins to chromatin.
    [Show full text]
  • FANCJ Regulates the Stability of FANCD2/FANCI Proteins and Protects Them from Proteasome and Caspase-3 Dependent Degradation
    FANCJ regulates the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation Komaraiah Palle, Ph.D. (Kumar) Assistant Professor of Oncologic Sciences Abraham Mitchell Cancer Research Scholar Mitchell Cancer Institute University of South Alabama Outline • Fanconi anemia (FA) pathway • Role of FA pathway in Genome maintenance • FANCJ and FANCD2 functional relationship • FANCJ-mediated DDR in response to Fork-stalling Fanconi Anemia • Rare, inherited blood disorder. • 1:130,000 births Guido Fanconi 1892-1979 • Affects men and women equally. • Affects all racial and ethnic groups – higher incidence in Ashkenazi Jews and Afrikaners Birth Defects Fanconi anemia pathway • FA is a rare chromosome instability syndrome • Autosomal recessive disorder (or X-linked) • Developmental abnormalities • 17 complementation groups identified to date • FA pathway is involved in DNA repair • Increased cancer susceptibility - many patients develop AML - in adults solid tumors Fanconi Anemia is an aplastic anemia FA patients are prone to multiple types of solid tumors • Increased incidence and earlier onset cancers: oral cavity, GI and genital and reproductive tract head and neck breast esophagus skin liver brain Why? FA is a DNA repair disorder • FA caused by mutations in 17 genes: FANCA FANCF FANCM FANCB FANCG/XRCC9 FANCN/PALB2 FANCC FANCI RAD51C/FANCO FANCD1/BRCA2 FANCJ SLX4/FANCP FANCD2 FANCL ERCC2/XPF/FANCQ FANCE BRCA1/FANCS • FA genes function in DNA repair processes • FA patient cells are highly sensitive
    [Show full text]
  • Fanconi Anemia, Bloom Syndrome and Breast Cancer
    A multiprotein complex in DNA damage response network of Fanconi anemia, Bloom syndrome and Breast cancer Weidong Wang Lab of Genetics, NIA A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia Bloom Syndrome . Genomic Instability: -sister-chromatid exchange . Cancer predisposition . Mutation in BLM, a RecQ DNA Helicase . BLM participates in: HR-dependent DSB repair Recovery of stalled replication forks . BLM works with Topo IIIa and RMI to Suppress crossover recombination Courtesy of Dr. Ian Hickson A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia P I l o r t n o BLM IP kDa C HeLa BLAP 250 Nuclear Extract 200- BLM* FANCA* 116- TOPO IIIα* 97- BLAP 100 MLH1* BLM IP BLAP 75 * 66- RPA 70 IgG H 45- * 30- RPA32 IgG L 20- * 12- RPA14 Meetei et al. MCB 2003 A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia P I A C N A F BLM IP HeLa FANCM= FAAP 250 BLAP 250 Nuclear Extract BLM* BLM* * FANCA* FANCA TOPO IIIα* TOPO IIIα* FAAP 100 BLAP 100 FANCB= FAAP 95 MLH1 FANCA IP BLM IP BLAP 75 BLAP 75 RPA70*/FANCG* RPA 70* FANCC*/FANCE* IgG H FANCL= FAAP 43 FANCF* RPA32* IgG L Meetei et al. MCB 2003 Meetei et al. Nat Genet. 2003, 2004, 2005 BRAFT-a Multisubunit Machine that Maintains Genome Stability and is defective in Fanconi anemia and Bloom syndrome BRAFT Super-complex Fanconi Anemia Bloom Syndrome Core Complex Complex 12 polypeptides 7 polypeptides FANCA BLM Helicase (HJ, fork, D-loop), fork FANCC regression, dHJ dissolution Topo IIIα Topoisomerase, FANCE dHJ dissolution FANCF BLAP75 RMI1 FANCG Stimulates dHJ dissolution.
    [Show full text]