Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL

Total Page:16

File Type:pdf, Size:1020Kb

Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL University of Rhode Island DigitalCommons@URI Senior Honors Projects Honors Program at the University of Rhode Island 2014 Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL proteins Joseph McClanaghan University of Rhode Island, [email protected] Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License. Follow this and additional works at: http://digitalcommons.uri.edu/srhonorsprog Part of the Biochemistry Commons, Biology Commons, Cancer Biology Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation McClanaghan, Joseph, "Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL proteins" (2014). Senior Honors Projects. Paper 367. http://digitalcommons.uri.edu/srhonorsprog/367http://digitalcommons.uri.edu/srhonorsprog/367 This Article is brought to you for free and open access by the Honors Program at the University of Rhode Island at DigitalCommons@URI. It has been accepted for inclusion in Senior Honors Projects by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL proteins Joseph D. McClanaghan Karissa L. Paquin, Paul A. Azzinaro; Faculty Sponsor Dr. Niall G. Howlett Department of Cell and Molecular Biology, University of Rhode Island, Kingston RI 02881 Background A pCMV- S1 FLAG/HA pLenti6.2- HPV16 E7 HA-FANCL Fanconi anemia (FA) is a rare X-linked and autosomal disease CUE COS7 + characterized by bone marrow failure, congenital defects, and S1/S2 DNA (ug) ND 1 2 6 8 increased cancer susceptibility. FA is caused by mutations in S2 any 1 of 16 genes. The protein products of these genes work S2/S3 together in the FA-BRCA DNA damage response pathway to S3 repair DNA interstrand crosslinks (ICLs). The FA-BRCA FANCL HBD pathway is a critical tumor suppressor pathway. α-HA.11 Chromodomain S4 HPV16 E7 B V5 FANCD2 α-Tubulin Tubulin V5 FANCD2 S1 1 2 3 4 5 V5 FANCD2 S2 Figure 6. Expression of pLenti6.2 HA-FANCL. COS7 cells were V5 FANCD2 S3 transfected with pLenti6.2-HA-FANCL, a FLAG/HA HPV16 V5 FANCD2 S4 control, or no DNA (ND). Cells were harvested and lysed in 2% Figure 3. Generation of overlapping fragments of FANCD2. SDS lysis buffer and analyzed via Western blotting using anti- Four FANCD2 fragments were generated, each with a N- HA and anti-tubulin antibodies. terminal V5 epitope tag. The four fragments are based on the Input IP: V5 Agarose four solenoids of FANCD2, designated S1-S4. COS7 + D2-S1 D2-S2 D2-S3 D2-S4 DNA (ug) ND 1 2 4 8 1 2 1 2 D2S3 COS7 + α-V5 α-D2 E35 FAND2-Ub D2S1 FAND2 Figure 1. Schematic model of the FA-BRCA pathway. Upon ICL D2S2 formation, the FA core complex monoubiquitinates FANCD2 D2S4 and FANCI. Upon monoubiquitination FANCD2 and FANCI α-Tubulin Tubulin α-HA.11 localize to sites of DNA damage where they interact with FANCL known DNA repair proteins. 1 2 3 4 5 6 7 8 9 Figure 4. Expression of pcDNA3.1-V5-FANCD2 Fragments. 1 2 3 4 5 6 7 8 COS7 cells were transfected with pcDNA3.1-V5-FANCD2 Figure 7. Co-Immunoprecipitation of V5-FANCD2 and HA- A B Substrate Ub D2 Ub fragments or no DNA (ND). Cells were harvested and lysed in FANCL. A co-IP was performed to determine if FANCD2 and 2% SDS lysis buffer, and analyzed via Western blotting using FANCL interact. COS7 cells were transfected with no DNA F-Box UBC UBE2T E Protein L anti-V5 and anti-tubulin antibodies. (ND; lanes 3 and 7), pcDNA3.1-V5-FANCD2 (V5-D2; lanes 1 and RBX1 pCMV- 5), pLenti6.2-HA-FANCL (HA-L; lanes 2 and 6), or both (V5-D2 + Skp1 C FLAG/HA pEAK8- HA-L; lanes 4 and 8). Samples were lysed in low stringency Cullin A COS7 + HPV16 E7 FLAG-FANCE buffer and immune complexes isolated with anti-V5 agarose. DNA (ug) ND 1 1 2 3 Samples were eluted and analyzed using Western blotting with anti-HA and anti-FANCD2 antibodies. SCF E2/E3 FA-BRCA E2/E3 Complex Complex α-FLAG FANCE Conclusions We have successfully generated mammalian expression Figure 2. Comparison of SCF E2/E3 and the FA-BRCA E2/E3 constructs for four overlapping V5-tagged FANCD2 fragments ubiquitin ligase complexes. The SCF (for Skp1-Cul1-F-box α-Tubulin Tubulin and HA-tagged FANCL. We have also successfully optimized protein) E3 ubiquitin ligase (A) is a large complex made up of conditions for their ectopic expression in COS7 cells and also the RBX1 E3 ubiquitin ligase, UBC E2 ubiquitin-conjugating 1 2 3 4 5 optimized the FLAG-tagged FANCE construct. In addition, we enzyme, and the F-box protein which recognizes the substrate. Figure 5. Expression of FLAG-FANCE. A FLAG-tagged FANCE have begun to perform co-IP experiments to determine the Here, we propose a similar model for the ubiquitination of construct was also obtained. COS7 cells were transfected with specific regions of FANCD2 to which FANCL and FANCE bind. FANCD2 by the FA core complex (B), where FANCL is the E3 pEAK8-FLAG-FANCE or no DNA (ND). Cells were harvested Mapping these sites of interaction will generate important ligase, UBE2T is the E2 ubiquitin-conjugating enzyme, and and lysed in 2% SDS lysis buffer, and analyzed via Western mechanistic insight into the regulation of this critical tumor FANCE is the substrate (FANCD2) recognition protein. blotting using anti-FLAG and anti-tubulin antibodies. suppressor pathway. This work was supported by grants from the NIH/NHLBI, the Department of Defense, and the University of Rhode Island Undergraduate Research Initiative .
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]
  • 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]
  • 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]
  • HEREDITARY CANCER PANELS Part I
    Pathology and Laboratory Medicine Clinic Building, K6, Core Lab, E-655 2799 W. Grand Blvd. HEREDITARY CANCER PANELS Detroit, MI 48202 855.916.4DNA (4362) Part I- REQUISITION Required Patient Information Ordering Physician Information Name: _________________________________________________ Gender: M F Name: _____________________________________________________________ MRN: _________________________ DOB: _______MM / _______DD / _______YYYY Address: ___________________________________________________________ ICD10 Code(s): _________________/_________________/_________________ City: _______________________________ State: ________ Zip: __________ ICD-10 Codes are required for billing. When ordering tests for which reimbursement will be sought, order only those tests that are medically necessary for the diagnosis and treatment of the patient. Phone: _________________________ Fax: ___________________________ Billing & Collection Information NPI: _____________________________________ Patient Demographic/Billing/Insurance Form is required to be submitted with this form. Most genetic testing requires insurance prior authorization. Due to high insurance deductibles and member policy benefits, patients may elect to self-pay. Call for more information (855.916.4362) Bill Client or Institution Client Name: ______________________________________________________ Client Code/Number: _____________ Bill Insurance Prior authorization or reference number: __________________________________________ Patient Self-Pay Call for pricing and payment options Toll
    [Show full text]
  • Complex Interacts with WRN and Is Crucial to Regulate Its Response to Replication Fork Stalling
    Oncogene (2012) 31, 2809–2823 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The RAD9–RAD1–HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling P Pichierri, S Nicolai, L Cignolo1, M Bignami and A Franchitto Department of Environment and Primary Prevention, Istituto Superiore di Sanita`, Rome, Italy The WRN protein belongs to the RecQ family of DNA preference toward substrates that mimic structures helicases and is implicated in replication fork restart, but associated with stalled replication forks (Brosh et al., how its function is regulated remains unknown. We show 2002; Machwe et al., 2006) and WS cells exhibit that WRN interacts with the 9.1.1 complex, one of enhanced instability at common fragile sites, chromo- the central factors of the replication checkpoint. This somal regions especially prone to replication fork interaction is mediated by the binding of the RAD1 stalling (Pirzio et al., 2008). How WRN favors recovery subunit to the N-terminal region of WRN and is of stalled forks and prevents DNA breakage upon instrumental for WRN relocalization in nuclear foci and replication perturbation is not fully understood. It has its phosphorylation in response to replication arrest. We been suggested that WRN might facilitate replication also find that ATR-dependent WRN phosphorylation restart by either promoting recombination or processing depends on TopBP1, which is recruited by the 9.1.1 intermediates at stalled forks in a way that counteracts complex in response to replication arrest. Finally, we unscheduled recombination (Franchitto and Pichierri, provide evidence for a cooperation between WRN and 2004; Pichierri, 2007; Sidorova, 2008).
    [Show full text]
  • Neurodegeneration in Accelerated Aging
    DOCTOR OF MEDICAL SCIENCE DANISH MEDICAL JOURNAL Neurodegeneration in Accelerated Aging Morten Scheibye-Knudsen This review has been accepted as a thesis together with 7 previously published pa- pers by the University of Copenhagen, October 16, 2014 and defended on January 14, 2016 Official opponents: Alexander Bürkle, University of Konstanz Lars Eide, University of Oslo Correspondence: Center for Healthy Aging, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen E-mail: [email protected] Dan Med J 2016;63(11):B5308 INTRODUCTION The global elderly population has been progressively increasing throughout the 20th century and this growth is projected to per- sist into the late 21st century resulting in 20% of the total world population being aged 65 or more by the year 2100 (Figure 1). 80% of the total cost of health care is accrued after 40 years of Figure 2. The phenotype of human aging. age where chronic diseases become prevalent [1, 2]. With an ex- that appear to regulate the aging process [4,5]. These include the ponential increase in health care costs, it follows that the chronic insulin and IGF-1 signaling cascades [4], protein synthesis and diseases that accumulate in an aging population poses a serious quality control [6], regulation of cell proliferation through factors socioeconomic problem. Finding treatments to age related dis- such as mTOR [7], stem cell maintenance 8 as well as mitochon- eases, therefore becomes increasingly more pertinent as the pop- drial preservation [9]. Most of these pathways are conserved ulation ages. Even more so since there appears to be a continu- through evolution and appear to regulate aging in many lower or- ous increase in the prevalence of chronic diseases in the aging ganisms.
    [Show full text]
  • Evolutionary Genomics of Human Intellectual Disability Bernard Crespi,1 Kyle Summers2 and Steve Dorus3
    Evolutionary Applications ISSN 1752-4571 ORIGINAL ARTICLE Evolutionary genomics of human intellectual disability Bernard Crespi,1 Kyle Summers2 and Steve Dorus3 1 Department of Biosciences, Simon Fraser University, Burnaby, BC, Canada 2 Department of Biology, East Carolina University, Greenville, NC, USA 3 Department of Biology and Biochemistry, University of Bath, Bath, UK Keywords Abstract genetic, genomic, intellectual disability, positive selection. Previous studies have postulated that X-linked and autosomal genes underlying human intellectual disability may have also mediated the evolution of human Correspondence cognition. We have conducted the first comprehensive assessment of the extent Dr Bernard Crespi, Department of Biological and patterns of positive Darwinian selection on intellectual disability genes in Sciences, 8888 University Drive, Simon Fraser humans. We report three main findings. First, as noted in some previous University, Burnaby, BC V5A 1S6, Canada. reports, intellectual disability genes with primary functions in the central ner- Tel.: 778 782-3533; fax: 778 782-3496; e-mail: [email protected] vous system exhibit a significant concentration to the X chromosome. Second, there was no evidence for a higher incidence of recent positive selection on Received: 20 July 2009 X-linked than autosomal intellectual disability genes, nor was there a higher Accepted: 28 July 2009 incidence of selection on such genes overall, compared to sets of control genes. First published online: 7 September 2009 However, the X-linked intellectual disability genes inferred to be subject to recent positive selection were concentrated in the Rho GTP-ase pathway, a key doi:10.1111/j.1752-4571.2009.00098.x signaling pathway in neural development and function.
    [Show full text]
  • Reduced FANCD2 Influences Spontaneous SCE and RAD51 Foci
    Oncogene (2013) 32, 5338–5346 OPEN & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia P Gravells1,3, L Hoh1,2,4, S Solovieva1, A Patil1, E Dudziec1, IG Rennie2, K Sisley2 and HE Bryant1 Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double- strand
    [Show full text]
  • Fanconi Anemia D2 Protein Confers Chemoresistance in Response to the Anticancer Agent, Irofulven
    3153 Fanconi anemia D2 protein confers chemoresistance in response to the anticancer agent, irofulven Yutian Wang,1 Timothy Wiltshire,2 Jamie Senft,1 Introduction 3 1,2 Sharon L. Wenger, Eddie Reed, Fanconi anemia is a genetic cancer-susceptibility syn- and Weixin Wang1,2 drome characterized by congenital abnormalities, bone marrow failure, and cellular sensitivity to DNA cross- 1 2 Mary Babb Randolph Cancer Center, Department of linking agents (1, 2). There are at least 12 Fanconi anemia Microbiology, Immunology, and Cell Biology, and 3Department of Pathology, West Virginia University School of Medicine, complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, Morgantown, West Virginia and M), and 10 Fanconi anemia genes (A, C, D1/BRCA2, D2, E, F, G, J, L, and M) have been cloned (1–8). All of the Fanconi anemia proteins function in a common path- Abstract way. Fanconi anemia proteins (A, B, C, E, F, G, L, and M) The Fanconi anemia-BRCA pathway of genes are fre- assemble in a nuclear complex that is required for mono- quently mutated or epigenetically repressed in human ubiquitination/activation of the downstream FANCD2 cancer. The proteins of this pathway play pivotal roles in protein (1, 2, 4, 6, 9–13). FANCL has been shown to DNA damage signaling and repair. Irofulven is one of a possess the E3 ubiquitin ligase activity to monoubiqui- new class of anticancer agents that are analogues of tinate FANCD2at Lys 561 (11). The deubiquitinating mushroom-derived illudin toxins. Preclinical studies and enzyme of FANCD2, USP1, has also been recently clinical trials have shown that irofulven is effective identified (14).
    [Show full text]