The Fanconi anemia pathway of genomic maintenance PDF
Preview The Fanconi anemia pathway of genomic maintenance
CellularOncology28(2006)3–29 3 IOSPress Review The Fanconi anemia pathway of genomic maintenance ∗ MariekeLevitus,HansJoenjeandJohanP.deWinter DivisionofClinicalGeneticsandHumanGenetics,VUMedicalCenter,vanderBoechorststraat7,1081BT, Amsterdam,TheNetherlands Abstract.Fanconianemia(FA),arecessivesyndromewithbothautosomalandX-linkedinheritance,featuresdiverseclinical symptoms,suchasprogressivebonemarrowfailure,hypersensitivitytoDNAcross-linkingagents,chromosomalinstabilityand susceptibility to cancer. At least 12 genetic subtypes have been described (FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all exceptFA-Ihavebeenlinkedtoadistinctgene.MostFAproteinsformacomplexthatactivatestheFANCD2proteinviamo- noubiquitination,whileFANCJandFANCD1/BRCA2functiondownstreamofthisstep.TheFAproteinstypicallylackfunc- tionaldomains,exceptforFANCJ/BRIP1andFANCM,whichareDNAhelicases,andFANCL,whichisprobablyanE3ubiq- uitinconjugatingenzyme.Basedonthehypersensitivitytocross-linkingagents,theFAproteinsarethoughttofunctioninthe repairofDNAinterstrandcross-links,whichblocktheprogressionofDNAreplicationforks.Herewepresentahypothetical model,whichnotonlydescribestheassemblyoftheFApathway,butalsopositionsthispathwayinthebroadercontextofDNA cross-linkrepair.Finally,thepossiblerolefortheFApathway,inparticularFANCFandFANCB,intheoriginofsporadiccancer isdiscussed. Keywords:Fanconianemia,cancer,geneticpredisposition 1. Carcinogenesis proto-oncogenes. These proto-oncogenes, which are highlyconservedinevolution,areimportantregulators Cancerarisesbymultiplealterationsinthegenome. ofnormalcellproliferationanddifferentiation.Whena Twogeneralclassesofgenesmaybedistinguishedac- mutationaltersthestructureoftheseproto-oncogenes, cording to their role in cancer formation: gatekeeper thereby making them constitutively active or altering genesandcaretakergenes.Thefirstclassiscomprised their expression levels or sites of expression, these of two types of genes: oncogenes and tumor suppres- proto-oncogenes become oncogenes. Oncogenes are sorgenes,whicharedirectlyinvolvedinthecontrolof responsible for uncontrolled cell growth by increased cellular proliferation. Control of cellular proliferation cell birth. They perform the same function as proto- canbeachievedbytwoseparatemechanisms,byreg- oncogenes but are beyond control or out of control. ulating cell proliferation and by regulating the occur- Justasproto-oncogenesarethoughttoregulatenormal rence of cell death. In normal tissues the rate of cell cellproliferation,theactionoftumorsuppressorgenes birth equals the rate of cell death, resulting in home- is to constrain cell growth in normal cells. Mutations ostasis.Previously,tumordevelopmentwasthoughtto thatinactivatetumorsuppressorgenesthereforeliber- be predominantly caused by an increased rate of cell atethecellfrom growthconstraintsimposedbythese birth. Today, it is recognized that tumors arise due to genes,whichnormallycontrolcelldeathorcellcycle animbalancebetweencellbirthandcelldeath.Genes arrest.Acombinationofactivatedoncogenesandinac- that promote cell birth or cell proliferation are called tivatedtumorsuppressorgenesgenerallycharacterizes acancercell. *Correspondingauthor:JohanP.deWinter,DivisionofClinical The second class of genes involved in cancer for- GeneticsandHumanGenetics,VUMedicalCenter,VanderBoe- mation are the caretaker genes or stability genes. chorststraat 7, 1081 BT Amsterdam, The Netherlands. Tel.: +31 204448283;Fax:+31204448285;E-mail:[email protected]. These genes are not directly involved in control of 1570-5870/06/$17.002006–IOSPressandtheauthors.Allrightsreserved 4 M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance cell growth, but are responsible for maintaining the 2. Fanconianemia integrity of the genetic information in the cell. DNA can become damaged by both endogenous as well as 2.1. Clinicalcharacteristics exogenous factors. Each cell has a number of differ- ent pathways that repair specific types of DNA dam- FA is a rare recessive disease with both autosomal age.Forexample,thenucleotideexcisionrepair(NER) and X-linked inheritance and has an estimated het- pathway is involved in repairing damaged bases such erozygouscarrierfrequencyofaround1in300,based as thymine dimers induced by UV light [41]. Inacti- on the incidence of affected individuals in the State vation of these DNA repair genes will result in loss of New York in 1971 [95,129,138]. The incidence of ofaneffectiveDNArepairsystem,whichmayleadto FA varies among ethnic populations. In some popu- ahighermutationrate.Inotherwords,stabilitygenes lations the incidence is higher due to a founder ef- keepgeneticalterationstoaminimumandwheninacti- fect or where consanguineous marriages are common vated,cellsbecomegeneticallyunstable,i.e.,genomic [122,151]. mutationswilloccuratahigherrate.Sincecancerde- Fanconi anemia was first reported in 1927 by the velopmentrequiresmultiplemutations,ahighermuta- SwisspaediatricianGuidoFanconi[37].Hedescribed tionrateisanimportantprerequisiteincancerdevelop- a family with three male children with pancytopenia ment.Importanttargetsformutagenesisaregatekeeper and birth defects. Diagnosis of FA patients has been genes (proto-oncogenes and tumor suppressor genes). basedonthisdescriptionformanyyears.Subsequently Current understanding of the process of carcinogene- FA became known as an autosomally inherited dis- sis is in line with the hypothesis that a cell needs to ease with diverse clinical symptoms, such as skele- acquire a high mutation rate or mutator phenotype in tal malformations (hypoplasia of the thumbs and ra- order to become at risk for developing into a malig- dial hypoplasia), hyper- pigmentation in the form of nantcancercell[91].Inthisprocesscellsnotonlyac- café-au-lait spots and/or areas of hypo- pigmentation, quire mutations, but also gain and lose chromosomes smallstatureandlowbirthweight(sometimesrelated andchromosomalregionsresultinginaneuploidyand to growth hormone deficiency) [156]. Renal abnor- chromosomaldisorganization[35]. malities are present in about one third of the FA pa- Inherited mutations in stability genes may lead to tients and include renal aplasia or hypoplasia, horse- two types of apparent modes of inheritance. In dom- shoe kidneys or double ureters. Urogenital malfor- inantly inherited conditions only one mutated gene is mations are also common, that is undescended testes inheritedandtheotherallelemustbeinactivatedbefore in males and uterine abnormalities in females; addi- an accelerated mutation rate is achieved (BRCA1/2, tionally, hypogonadism and infertility are common in for predisposition for breast/ovary cancer, and DNA both [146]. The most important clinical features of mismatch repair genes, for hereditary nonpolyposis FA are in fact haematological, as FA is a bone mar- colorectal cancer). In recessively inherited diseases row failure syndrome. FA patients show a high inci- (such as xeroderma pigmentosum, Bloom syndrome, dence of aplastic anemia, myelodysplastic syndrome andFanconianemia),bothmutatedgenesareinherited (MDS) and acute myeloid leukemia (AML). At birth fromtheparentsandallthecellsofthebodyaregeneti- thebloodcountsareusuallynormal,thefirstsymptom callyunstable.Thereforeinthesesyndromestheacqui- of bone marrow failure being macrocytosis, followed sitionofadditionalmutationsinoncogenesandtumor by thrombocytopenia and neutropenia [18,146]. Pan- suppressor genes is much more likely. After a muta- cytopenia usually does not set in until about 7 years tioninanoncogeneortumorsuppressorgene,thecell of age [9,18]. The majority of the patients eventually startstoproliferateabnormallyandhasbecomeprone dieofAML,forwhichtheyhaveastronglyincreased todevelopintoacancercell. risk (about 1000 times) [121]. Those patients that do Accordingly, a germline mutation in any of these survive the haematological malignancies have to deal geneswillleadtocancerpredisposition,thatis,cancer with an array of solid tumors, mainly squamous cell ariseswithhighprobabilityandoftenmultipletumors carcinomasoftheheadandneckarea,for whichthey will develop at an earlier age than in individuals who show a similarly increased risk [5,6]. Bone marrow develop sporadic tumors [152,153]. In this review we failure in combination with cancer proneness reduces willfocusonthestabilitygenesthatunderlytheinher- the average life expectancy to about 15 to 20 years itedsyndromeFanconianemia. [24,153]. M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance 5 2.2. Cellularcharacteristics 2.4. Cellcycleabnormalities The process of cell division is tightly regulated by The first major discovery characterizing FA at the cell cycle checkpoints, since an abnormal cell cycle cellular level was that FA lymphocytes exhibit exces- mayleadtomalfunctioningdaughtercellsanduncon- sive spontaneous chromosomal instability. This insta- trolledgrowth.AttheG1cellcyclecheckpoint,thecell bility was seen in the form of chromatid gaps and determineswhetherallthepreparativestagesforDNA breaks, and various chromatid interchanges. Such in- synthesis have been completed correctly and whether terchanges involve heterologous chromosomes thecellisreadytoproceedintotheSphase.Duringthe [127,128].Thischromosomalinstabilityfeatureisnow DNAreplicationphaseanSphasecheckpointensures thoughttoberesponsiblefor thecancersusceptibility thatDNAsynthesiscanbesuccessfullycompleted,be- ofFApatients. forecellscanprogresstotheG2phase.IfDNAdam- agehasoccurredsuchdamagewillhavetoberepaired 2.3. Cross-linkerhypersensitivity before the cellcanproceedinto theG2 phase. This is ensured by this checkpoint, although certain types of DNAdamagecanalsoberepairedduringtheG2phase. Anotherimportantdiscoveryhasbeenthehypersen- TheG2/Mcheckpointensuresthatthecellisprepared sitivity of FA cells to the clastogenic effect of cross- andreadyforcelldivisionintheMphase.Ifcellsare linking agents, such as mitomycin C, diepoxybutane delayedorarrestedatacertainpointinthecellcycle, andcisplatin[10,62,126,161].Suchbifunctionalalky- this will endure for as long as necessary to repair all latingagentsgenerateDNAcross-linkdamage,which thedamage.Onlythenwillthecellsbeabletopassthe greatly exacerbates the chromosomal instability typi- checkpoint.Ifthedamagecannotberepaired,thecells callyseeninFAcells.Thisspecifictraitisnowwidely maygointoapoptosis. used in the diagnosis of FA patients. Kano and Fuji- FAcellsgenerallyexhibitanabnormalityincellcy- wara (1981) claimed that FA cells also show an in- cledistributioninthattheyshowanincreasedpropor- crease in the formation of sister chromatid exchanges tionofcellswith4NDNAcontent.Thisindicatesthat (SCE)afterMMCtreatment,suggestingaroleforthe inFAcellsaG2/MorlateSphasedelayorarrestoc- FApathwayinhomologousrecombinationrepair[71]. curs[36,77].ThisprolongedG2phaseorG2cellcycle However,morerecentevidenceshowsthatSCEforma- arrest is further enhanced after treatment with cross- tioninFAcellsissimilarasinnormalcellsafterMMC linking agents [68]. Since p53, a well known tumour treatment (Joenje and Oostra, unpublished data) cast- suppressor is involved in G1/S and G2/M cell cycle ingdoubtovertheoriginaldatabyKanoandFujiwara. transitions, FA cells may be abnormal through a di- Cross-linking agents or bifunctional alkylating rectinteractionbetweenanFAproteinandp53.How- agentsgeneratepredominantlyintrastrandcross-links, ever, FA cells show a normal p53 protein induction whichconnectbaseswithinthesamestrand.However, after treatment with MMC or other DNA damaging the same agents also generate interstrand cross-links, agents. Moreover, p53-dependent apoptosis is normal in which the antiparallel DNA strands in a double in FA cells following treatment with MMC. Further- helix are connected [6]. Interstrand cross-links repre- more, FA cells and normal cells behave similarly in sent only a small fraction of the lesions formed by cell cycle progression from G1 phase to S phase [74, DNAcross-linkingagents(5–13%forMMC),butare 77,79]. Taken together, these results suggest that p53 thought to be the most toxic lesion, since they inhibit inductionandG1/StransitionisnormalinFAcellsand DNAstrandseparationandthereforeblockDNArepli- G2/Marrestdoesnotoccurthroughadirectinteraction cation, transcription and segregation [32,159]. The betweenanFAproteinandp53. sensitivity seen in FA cells seems to be restricted to Studies have shown that the type of damage that interstrandcross-links,astheydonotshowsensitivity is induced determines the type of repair that is called to UVC, a typical intrastrand cross-linker, monofunc- uponandatwhichstageofthecellcyclerepairisoc- tionalalkylatingagentssuchasEMS,ortoionizingra- curring. Gamma-irradiation during G2 causes double diation[34,42,69,161].However,thereissomedispute strand breaks (DSB), which causes a G2/M cell cy- aboutthelatteragent,sincesomepapersdoreporthy- clearrest[57].However,inductionofinterstrandcross- persensitivityofFAcellstoionizingradiation[15,21, linksduringG2stageofthecellcycledoesnotresultin 47,139]. chromosomalbreakageoraG2/Marrestuntilthenext 6 M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance cell cycle. The same holds true for interstrand cross- linksinducedduringtheG1stage,whichdoesnotre- sult in a G1/S cell cycle arrest. In both FA and wild type cells chromosomal breakage and a cell cycle ar- rest is only observed, after the cells have undergone DNAreplication[2,3].ThisindicatesthatDNArepli- cation is required in wild type and FA cells to induce a G2/M arrest and chromosomal breakage. Since ar- restedFAcellshavea 4NDNAcontent,it isunlikely thattheFApathwayisinvolvedinacheckpointthatin- ducesearly-ormidSphasearrest.Morelikelyisthat the G2 arrest observed in FA cells represents incom- plete DNA replication and an arrest in late S-phase, Fig.1.RelativeprevalencesofcomplementationgroupsinFA.Re- ratherthanaG2/Marrest.Incontrasttowildtypecells, sultsarebasedonthefirst248FAfamiliesclassifiedbytheEuro- FAcellstake upto threetimes longertoclear this ar- peanFanconiAnemiaResearchProgram(1994–2003).Thisnumber rest, indicating that the FA proteins are somehow in- includesthereferencecelllinesforgroupsA,B,C,D1,andD2.The absolutenumberspergroupwereasfollows:A:159;B:4;C:23;D1: volved in resolving interstrand cross-links during late 8;D2:8;E:6;F:5;G:21;I:4;J:8;L:1,M:1.Figurereproduced, S-phase [2]. Furthermore, FA cells do not stop DNA withpermission,fromthethesisofM.Levitus. replicationafterinterstrandcross-linksareinduced,as isseeninwildtypecells[22,124]. al.in1985,Strathdeeetal.in1992andfurtherdevel- Overall,itseemsthatFAcellshaveadefectduring opedbyJoenjeetal.in1995[33,65,137].Celllinesare DNAreplicationintheSphaseofthecellcycle,either fusedpairwisetoresultinheterokaryons,includingvi- duetodefectiverepairofDNAdamageinducedduring ablehybridsthatpossess4NDNAcontent.Thisfusion theS-phaseor todamage-resistantDNAsynthesis,or hybridis thentestedfor MMC sensitivityinagrowth both, which ultimately results in a late S-phase delay inhibition assay. If the fusion partner cell lines were ratherthanaG2orG2/Mdelay. defectiveinthesamegene,thehybridisstillsensitive and the partners are said to belong to the same com- plementationgroup.If,however,bothcelllinesarede- 3. GeneticbasisofFanconianemia fective in different genes, the defect will be corrected inthehybridtoresultinwildtypesensitivitytoMMC 3.1. HeterogeneityinFanconianemia and the cell lines are said to belong to different com- plementationgroups(Fig.2).Byapplyingthismethod Fanconi anemia is highly heterogenous, both clin- toalargenumberofFApatients,atotaloftencomple- ically and genetically. Fanconi anemia can be subdi- mentationgroupscouldbedefined(FA-AtoFA-J,in- videdinnolessthantwelvegeneticsubtypesorcom- cludingtheretractedFA-Hgroup).Althoughthispro- plementationgroups,FA-AtoFA-M[33,64–66,84,94, cedurehasbeenquiteeffectiveindefiningcomplemen- 96,136,137].Eachgroupisconnectedtoadistinctdis- tationgroupsforFA,theanalysishassomepitfalls. easegene.Notallgroupsareequallyrepresented.Most One of these is false non-complementation. In this patients belong to group FA-A (approximately 60– case a fusion hybrid shows a FA-like phenotype and 80%), followed by groups FA-C and FA-G. The four both cell lines are therefore classified as belonging to smallest groups are FA-L and FA-M with both only a the same complementation group. However, some fu- single patient identified so far, followed by the FA-I sion hybrids can lose chromosomes, which can lead andFA-Bgroupswithfourpatientspergroup(Fig.1). to misclassification if the complementing chromo- somesarelost.Therefore,non-complementedhybrids 3.2. Complementationanalysis shouldalwaysbetestedforchromosomelosstoavoid such misclassification. Although this minimizes the Complementation groups were defined by cell fu- chance of wrong classifications, this approach does sionstudiesandcomplementationanalysis.Inthispro- notcompletelyruleoutmisclassification,asillustrated cedure two immortalized FA patient lymphoblastoid by the analysis of patients in the FA-D group. This cell lines are created and marked with a selection group was composed of four patients and a textbook marker, as first described by Duckworth-Rysiecki et example of non-complementation of fully tetraploid M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance 7 Fig.2.ComplementationanalysisinFA.Iftwopatientcelllinesarefusedtogether,thehybridcellcaneitherbecomesensitive(S,inwhich50% growthreduction(IC50)isseenataconcentrationbelow10nMMM)orresistant(R,withanIC50valueofmorethan10nMofMMC).An MMC-sensitivecelllineindicatesthatbothpatientcelllinesaredefectiveinthesameFAgeneandthereforebelongtothesamecomplementation group(greenhybridcell).If,however,ahybridcellbecomesresistanttoMMC,theresultindicatesthatthecelllinesaredefectiveindifferent FAgenes,sothattheMMCsensitivityiscorrected(redhybridcell).(FigurereproducedwithpermissionfromNatureReviewsGenetics(Joenje andPatel)[67](cid:2)c (2001)MacmillanMagazinesLtd.) hybrids [64,65]. Eventually this single group turned The advantage of this method is that it is relatively out to be composed of two different complementa- quick, but is dependent on the availability of a suit- tion groups, with two cell lines mutated in one gene ableantibodytodetecttheprotein.Furthermore,some (FANCD1/BRCA2) and two cell lines mutated in an- pathogenicmissensemutationsstillallownormallev- othergene(FANCD2)[58,145].Theoppositecanalso elsoffull-lengthproteintobeproduced.Resultsfrom occur, that is “false” complementation. In this case a Western blot procedures are therefore often inconclu- fusion hybrid shows a wild type phenotype, normally sive, but could provide useful indications how to pro- indicatingthatbothcelllinesbelongtodifferentcom- ceed to make a final assignment. The ultimate way to plementationgroups.However,insomerarecasesboth classifyaFApatientisbyidentifyingpathogenicmu- cell lines may belong to the same complementation tationsinbothallelesofaFAgene.Currently,onlyfor group instead. This happened with the FA-H group theFA-Icomplementationgroupthediseasegenehas which was represented by a single cell line [66]. Fi- notbeenidentifiedyet. nally,thisFA-Hcelllineturnedouttohavemutations Identification of new FA genes has been accom- intheFANCAgeneandthereforebelongedtotheFA-A plished by complementation cloning, positional complementationgroup;asaresult,groupHwaswith- cloning or protein complex purification. Positional drawnandnew,morestringent,criteriaforthedefini- cloningisusuallybasedonlargefamiliesbelongingto tionofcomplementationgroupswereproposed[64]. the same complementation group. In the case of con- After the genes for some of the complementation sanguineousfamiliesfindingasinglehomozygousre- groups were identified, alternative methods became gion within the entire genome identical in all patients availabletoclassifyFApatientsaccordingtocomple- (homozygosity mapping) reveals the approximate lo- mentation group. One method involves the introduc- cation of the defective gene and screening candidate tionofacDNAintotheunclassifiedcellline.Thiscan genes within the region for mutations in the patients beaccomplishedbyinsertingthecDNAintoaretrovi- will finally identify it. However, by using microcell- ralorepisomalexpressionvectorandtransferintothe mediated chromosome transfer, a combination of po- cells by transduction or transfection methods. How- sitionalcloningandcomplementationcloning,achro- ever,thisprocedureisonlyusefulifthedefectivegene mosomeorpartofachromosomecanbeidentifiedhar- oftheparticularcomplementationgroupisknown.An- boringthedefectivegeneusingasinglecellline.Inthis otheralternativeistodetectthepresenceorabsenceof procedure,acompleteorpartialhumanchromosomeis theFAproteinofinterestviaaWesternblotprocedure. introduced into the FA cell line. This hybrid can then 8 M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance Fig.3.ComplementationcloninginFA.DifferentcDNAs,containedinalibraryderivedfromwildtypehumanlymphoblasts,areclonedinto thepREP4vector.ThisvectorisintroducedintoaMMC-sensitiveFAcellline.First,ahygromycinselectionisperformedtoselectforthose cellsthathavetakenupavector.Secondly,thiscellpopulationisselectedforthosecDNAsthatconferMMCresistance.ThisMMCresistant cellpopulationislikelytocontaintheFAgenedefectiveintheoriginalcellline.ThedifferentcDNAsinthispopulation,allcandidate-FAgenes, arethenextractedandexamined.DetectionofmutationsinacandidategeneinthepatientprovidesfinalproofthatanewFAgenehasbeen identified.(FigurereproducedwithpermissionfromNatureReviewsGenetics(JoenjeandPatel)[67](cid:2)c (2001)MacmillanMagazinesLtd.) betestedforcomplementationofitsMMCsensitivity. startedwiththediscoveryoftheFANCCgenein1992 Inthiswaythepresenceofacomplementingpieceof using complementation cloning (Fig. 3) [136]. Com- chromosomemaybecorrelatedwithcomplementation plementation cloning is also referred to as functional ofthecellulardefect.Assoonasthecriticalregionhas cloning,sinceitisbasedontheprinciplethatintroduc- beennarroweddownsufficiently,subsequentscreening tionofthewildtypevariantofthedefectivegenecom- ofcandidategenesintheregionwillrevealthedefec- plements the phenotypic defect in the cell line. This tivegene.Proteincomplexpurificationisbasedonthe phenotypic trait makes it possible to select for cells precipitationof FA (core complex) proteins and iden- thathavebeencomplementedandthereforephenotyp- tification of all interacting proteins. These interacting ically corrected. For FA, this phenotypical defect is proteins are candidate-new FA proteins and mutation theextremesensitivitytocross-linkingagents,suchas screening in patient cell lines may reveal whether a MMC.IntroducingacDNAexpressionlibraryintothe newFAgeneandconsequentlyanewFAcomplemen- cellsandsubsequentselectionforthosecellsthathave tationgrouphasbeenfound.Thevariousmethodsused takenupcDNAsandafinalselectiononMMCselects to identify FA genes are discussed in more detail be- for only those cells that are corrected for the cellular low. phenotype.Theseresistantcellsaretheoreticallycom- plementedwith the cDNAor gene that is defective in 3.3. CloningFAgenes the original cell line. Identification of that cDNA and mutation screening in the patient must prove if a new The general idea that each complementation group FAgenehasactuallybeenidentifiedornot. would be defective in a single disease gene is sup- The method seems quite straightforward but does portedbytheactualsituationsofar;presently,onlythe have some prerequisites: first, the cDNA library used FANCI gene has not been identified.FA gene cloning must contain the relevant cDNA with its open read- M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance 9 ingframeintact;second,theFAproteinshouldnotbe proximatemolecularweight.TheFANCLproteinwas toxic when overexpressed; third, the cell line should initially termed FAAP43, since it has a molecular be able to take up the DNA,which is often a limiting weightof43kDa.Westernblotexperimentsrevealeda factor,sincehumanlymphoblastoidcelllinesareusu- singleFApatientwhoselymphoblastsmissedFANCL ally reluctant to take up exogenous DNA; fourth, the protein expression. Mutation analysis then confirmed FA cell line should not revert spontaneously at a sig- that this patient was indeed mutated in the corre- nificantratetoaMMC-resistantstate.Inspiteofthese sponding gene, termed FANCL, so that a new FA limitationsanumberofgeneshavebeenclonedusing gene and complementation group were discovered at this method. After the initial identification of FANCC the same time. Next to FANCL, three other proteins this same approach was used to identify FANCA in were co-precipitated with FANCA, namely, FAAP95, 1996[89],althoughnexttoidentificationthroughcom- FAAP100,andFAAP250.Byusingthesameapproach plementationcloning,FANCAwasindependentlyiden- asusedtoidentifyFANCL,FAAP95wasidentifiedas tified by positional cloning as well [1]. Complemen- FANCB, while FAAP250 was identified as FANCM. tation cloning continued to be very successful in the ThisleftonlyFAAP100unidentified,whichcouldrep- years thereafter, when three more genes were identi- resentanewFAgeneandcomplementationgroup.The fied.FANCGwasfoundin1998,followedbyFANCF FA-LandFA-Mcomplementationgroupsaretheonly andFANCEin2000[28,29,31]. groups todatethat arerepresentedby a singlepatient FANCD2 and FANCJ/BRIP1 were cloned by a dif- andinwhichthegeneswerefoundbeforethecomple- ferentmethod.Forthesegenesacombinationofposi- mentationgroupswereidentified.Table1summarizes tionalandcomplementationcloningwasused,alabo- all currently known FA genes, how they were identi- riousbutratherrobustproceduretoidentifynewgenes. fied,theirchromosomallocations,andfeaturesoftheir First, microcell-mediated chromosome transfer iden- proteinproducts. tified chromosome 3 as the complementing chromo- someforFANCD2.Next,positionalcloningusingpar- tiallydeletedchromosomes3revealedamaplocation 4. ProteinsactingupstreamofFANCD2activation at3p22–26,followedbytheidentificationofthedefec- tive gene in this group by screening candidate genes 4.1. FANCA,FANCB,andFANCG for mutations [145,162]. FANCJ/BRIP1 was cloned using the same approach, but the other way around. FANCA was first found to interact with FANCC First,linkageanalysisrevealedchromosome17asthe using co-immunoprecipitation experiments [30,44,75, most likely chromosome to harbor the FANCJ gene, 78].AdirectinteractionbetweenFANCAandFANCC which was confirmed by microcell-mediated chromo- was not found using yeast two-hybrid analysis, indi- sometransfer.Next,FANCJ/BRIP1wasscreenedasa cating that FANCA and FANCC might be part of the candidategeneandwasfoundtocarrymutationsinall samecomplex,butdonotbinddirectlytooneanother FA-J patients examined [85]. FANCJ was also inde- [59,93]. pendentlyidentifiedusingslightlydifferentmethodol- A direct interaction was detected between FANCA ogy[86,88]. and FANCG by co-immunoprecipitation experiments FANCD1 was identified by the candidate gene ap- andyeasttwo-hybridanalysis[30,46,51,59,73,93,119, proach. Mutation screening of BRCA2 in FA-D1 pa- 154].ThisinteractionispresentinallFAcomplemen- tientsrevealedbiallelicpathogenicmutations,indicat- tationgroups,indicatingthatformationoftheFANCA- ingthatBRCA2isinfacttheFANCD1gene[58]. FANCG subcomplex is one of the initial steps in the For the last genes cloned so far, FANCL, FANCB, FAcore-complexassembly[30,154]. and FANCM, an approach other than complementa- FANCA has both a nuclear and a cytoplasmic lo- tion cloning or positional cloning was used [94–97]. calization. An N-terminal bipartite NLS sequence, in The identification of these genes was based on pro- combination with sequences at the C-terminus are teincomplexpurification.Byidentifyingproteinsthat needed to assure proper nuclear localization of the bindtotheFANCAprotein,thecommoncomplexpro- protein [78,87,89]. Based on the predominant cyto- teins FANCC, FANCG, and FANCF were found, in plasmiclocalizationofFANCAinFA-Bcellsandthe addition to a number of unidentified proteins. These lack of phosphorylated FANCA in cells of all com- were termed FAAPs, for Fanconi Anemia Associated plementation groups (except in FA-D1), it was long Proteins, followed by a number indicating their ap- thought that FANCB might be the kinase responsible 10 M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance Table1 CharacteristicsofallcurrentlyknownFAgenesandproducts Gene Identification Location CDSsize Proteinsize Motifsordomains method (bp) (kDa) FANCA CC/PC 16q24.3 4368 161 BipartiteNLSandLeucinezipper FANCB PP Xp22.31 2579 95 NLS FANCC CC 9q22.3 1674 65 – FANCD1/BRCA2 CS 13q12.3 10256 384 8BRCmotifsforRAD51binding FANCD2 PC 3p25.3 4415 155/162 MonoubiquitinationatK561 FANCE CC 6p21.2–21.3 1610 63 2NLS FANCF CC 11p15 1124 42 Adaptorprotein XRCC9/FANCG CC 9p13 1868 70 7TPRmotifsforproteinbinding FANCJ/BRIP1 PC,CS 17q22 3749 141 7helicasemotifs,ATPdependent 5(cid:1)→3(cid:1)helicase FANCL PP 2p16.1 1127 43 Ring finger motif typical for E3 ubiquitinligases FANCM PP 14q21.3 6044 250 Endonuclease and helicase do- main CC,complementationcloning;PC,positionalcloning;PP,proteinassociation;CS,candidategeneapproach. for nuclear accumulation of phosphorylated FANCA that the TPRs are important for the interaction with [30,166].However,FANCBdoesnotcontainaprotein FANCA [16]. The interaction between FANCA and kinase domain, making it very unlikely that FANCB FANCG is direct and based on binding of FANCG isresponsibleforFANCAphosphorylation.Addition- to the N-terminal NLS sequence of FANCA [45,76, ally,FANCAisnotphosphorylatedinallcomplemen- 154]. Apart from binding to the FANCA protein, the tation groups that affect the core complex and there- C-terminal part of FANCG is thought to bind the foreitseemsmorelikelythatthewholecorecomplex FANCC protein, since deletion of the last 39 amino isinvolvedinthismodificationorinstabilizationofthe acids makes FANCG unable to recruit FANCC into modifiedformofFANCA. the complex [76]. However, no direct interaction has In the absence of FANCB, FANCL is also not been found between FANCG and FANCC, although detected in the nuclear compartment. FANCB was theproteinsbelongtothesamecomplex[30,46,51,93]. found to directly interact with FANCL in the mam- ItseemsmorelikelythatFANCFformsthebridgebe- maliantwohybridassay,suggestingthatFANCBmay tween FANCG and FANCC since the C-terminus of aid transport of FANCL into the nucleus, through its FANCFbindsFANCGandtheN-terminusofFANCF NLS sequence (Medhurst et al., unpublished data). isabletobindFANCCandFANCE[83]. SinceFANCAisalsoneededforthenuclearaccumu- Apparently,FANCGalsobindsdirectlytoFANCD1/ lation of FANCL and the FANCB/FANCL subcom- BRCA2 at the N- and C-termini, around the BRC re- plex does bind FANCA, it is possible that FANCB peats[61].Thepreciseimplicationsofthisinteraction and FANCL enter the nucleus together with FANCA are not known at present, but combined with results andFANCG[94](Medhurstetal.,unpublisheddata). indicating a direct binding of FANCA and FANCJ to Alternatively, FANCA/FANCG and FANCB/FANCL BRCA1, FANCD2 to FANCD1/BRCA2, and the co- might enter the nucleus separately through their NLS localizationofFANCD2andBRCA1inDNAdamage- sequences and form a subcomplex inside the nucleus, inducible nuclear foci, these data strengthen the no- rather than in the cytoplasm. This larger complex is tionthattheFApathwayiscloselylinkedtotheBRCA thenneededtomaintainnuclearlocalization. pathway and possibly involved in repair through ho- FANCG, like FANCA, is found in the nucleus as mologousrecombination(HR)[38,47,85,158]. well as in the cytoplasm [154]. The protein has seven motifsknownastetratricopeptiderepeatmotifs(TPR), 4.2. FANCCandFANCE which are 34-nucleotide sequences thought to be in- volvedinprotein-proteininteractions.Disruptingthese Atfirst,FANCCwasthoughttohaveacytoplasmic motifs leads to reduced FANCA binding, suggesting function, due to its predominantly cytoplasmic loca- M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance 11 tion in cell fractionation and immunofluoresence ex- teractionofFANCCwithp53,possiblyimplicatingin- periments [165,168,169]. Later a small proportion of volvementinprocessesregulatedbyp53.Anotherpos- the protein was also detected in the nuclear compart- sibilityisthatthesemalignanciesmanifestmoreread- ment[55].NexttothedebatedFANCA/FANCCinter- ily when p53 control is disrupted. Recently, FANCC action, FANCC was also found to interact with other and p53 were both suggested to regulate G2 check- FAproteins,especiallywithFANCE,whichisadirect pointcontrol,againlinkingFANCCtocellcyclecon- interaction[51,93,111]. trol[39,40]. FANCE,apredominantlynuclearprotein,isthought topromotenuclearlocalizationofFANCC.Co-expre- 4.3. FANCF ssion of the FANCE protein with FANCC, promotes FANCC to be targeted into the nucleus. In the ab- sence of FANCE, lower levels of FANCC were seen FANCF shows homology to the prokaryotic RNA- incytoplasmandnucleus,whichcouldberestoredaf- binding protein ROM, which suggested a possible ter re-introduction of the FANCE protein. Addition- function for FANCF in RNA binding [29]. How- ally, C-terminal FANCC mutants are unable to inter- ever mutational disruption of the ROM motif does actwithFANCEandalsolosetheabilitytoco-localize not appear to alter FANCF’s functional activity in withFANCEinnuclearfoci[111,140]. a complementation assay [83]. Immunoprecipitation As FANCC presumably does not interact with the studies and cellular fractionation studies have pro- cytoplasmic complex of FANCA, -B, -G and -L and vided evidence for a mainly nuclear localization of since this protein lacks an apparent NLS sequence, FANCF, where it interacts with FANCA, FANCC, nuclear localization of FANCC might partly depend and FANCG [30]. In yeast two-hybrid assays a di- on its relatively small size, which through diffusion rectinteractionbetweenFANCFandFANCGisfound transportmayberesponsibleformovingFANCCinto through the C-terminal region of FANCF [51,83,93]. the nuclear compartment. However, nuclear localiza- The N-terminal part of FANCF is needed to bind tionofFANCCcouldalsobeduetoacarrierprotein, the FANCC/FANCE complex, although no direct in- for example FANCE, or a still unidentified NLS se- teraction between FANCF and FANCC, or FANCE quence. FANCE enhances localization of FANCC in alone, has been found. Also this region seems to thenucleus,possiblybybindingandretainingFANCC be necessary for stabilizing the FANCA/FANCG in- in the nucleus. If no FANCE is present, for example teraction by FANCF [83]. These direct interactions in FA-E cells, all FANCC resides in the cytoplasm of FANCF with FANCG and FANCC/FANCE and [51,55,111,140]. the indirect interaction with FANCA, may impli- FANCE has also been shown to interact with cate FANCF as a key protein in the assembly of a FANCA and FANCG in co-immunoprecipitation ex- large FA core complex in the nucleus, bringing to- periments [140]. However, only a weak direct inter- gether the FANCA/FANCG/FANCB/FANCL and the action of FANCE with FANCA and FANCG was de- FANCC/FANCEsubcomplexes. tectedbyyeasttwo-hybridanalysis,indicatingthatthis ispossiblyanindirectinteraction,whichisdependent 4.4. FANCL onotherproteins[93].Adirectinteractionbetweenthe C-terminusofFANCEandtheN-terminusofFANCD2 has been found, thereby linking FANCD2 to the FA This protein was identified by protein purification complex[51,111].FANCCalsointeractswithanum- with antibodies against FANCA. FANCL is present berofunrelatedproteins,suchascyclin-dependentki- bothinthecytoplasmandinthenucleus[88,94].Pre- nasecdc2,Hsp70andPKR.Thelattertwointeractions viously, it was assumed that BRCA1 with its RING suggest a role for FANCC in supporting hematopoi- finger motif was responsible for the monoubiquitina- etic cell survival, since Hsp70 suppresses PKR activ- tionofFANCD2[47].Thisideawassubsequentlydis- ity, which is involved in this process [112]. FANCC putedbecausesiRNAknock-downofBRCA1ormuta- has also been reported to be involved in apoptosis tionoftheRINGfingermotifsinBRCA1didnotaffect and tumorigenesis, since in FANCC/p53 double mu- monoubiquitinationofFANCD2[148].Later,withthe tantmice,malignanciesareformedmorerapidlythan findingofFANCLanditsRINGdomainitbecameevi- in thesingle mutantmiceandare of the sametype as dentthatFANCLismuchmorelikelytoberesponsible thosefoundinFApatients.Theseresultssuggestanin- formonoubiquitinationofFANCD2[94,98].Although 12 M.Levitusetal./TheFanconianemiapathwayofgenomicmaintenance it is logical to assume that FANCD2 and FANCL at 4.6. FAAP100 some stage engage in a direct interaction, no direct interaction has been detected so far. It is more likely FAAP100 (Fanconi Anemia Associated Protein of 100kDa)wasidentifiedbyproteinpurificationexperi- that FANCE is the missing link between FANCL and mentsusingantibodiesagainstFANCA,andwasfound FANCD2,bridgingtheE3ligasewithitssubstrate.The simultaneously with FANCL, FANCB and FANCM. onlydirectinteractionfoundforFANCListhebinding FAAP100 thus represents a candidate-new FA gene withFANCAandFANCB,whichseemstobeasimi- and complementation group. FAAP100 is not likely larbindingashasbeenobservedforFANCE,FANCC, to be encoded by the FANCI gene, since normal pro- and FANCF. FANCL and FANCB need to bind first tein expression in FA-I cells was found. So far, no beforetheycaninteractwithFANCA(Medhurstetal., FA patients have been found carrying mutations in unpublisheddata). FAAP100;untilthathappens,FAAP100willretainthe statusofan‘associatedprotein’[97]. 4.5. FANCM 4.7. FANCI FANCM,likeFANCLandFANCJ(seebelow),also Although the protein defective in the FA-I comple- has functional domains: a DEAH-box helicase do- mentation group is unknown so far, a clue about its main,whichseemstorenderanATP-dependentDNA function and position within the FA pathway may be translocase activity to FANCM, and an endonucle- deducedfromthecharacteristicsofFA-Icelllines.By asedomainhomologoustoERCC4/XPF.FANCMap- focusingontheinteractionsoftheFAproteinswithin pearstobethehumanorthologofarchaealDNArepair these cell lines, it has become evident that in FA-I cells the core complex is properly formed. This in- protein Hef, which is probably involved in process- dicatesthatFANCI,likeFANCD2,FANCD1/BRCA2 ing of stalled replication forks [96,102]. FANCM is and FANCJ, does not belong to this core complex post-translationallymodifiedinresponsetoDNAdam- and must function downstream or independent. How- age or replication block, which is not dependent on ever,FANCD2isnotmonoubiquitinatedinFA-Icells, the ubiquitin ligase activity of the core complex. Fur- suggesting that its function is upstream of FANCD2 thermore, FANCM becomes hyperphosphorylated af- activation, but downstream of the FA core complex. ter DNA damage, possibly by ATR, and is capable of FANCI may assist FANCL in the activation of enteringthenucleusonitsown.WithoutFANCM,the FANCD2. The reduced presence of FANCD2-S in initial interaction of FANCA and FANCG seems to nuclear extracts of FA-I cells suggest a function for be compromised, indicating that FANCM is involved FANCIinbindingFANCD2-Stothechromatin(Fig.4). in the initial steps of the FA core complex assem- AsbothformsofFANCD2seemtoasscociatewiththe bly.Additionally,FANCAandFANCLshowdeficient chromatin,itispossiblethatFANCD2ismonoubiqui- nuclear localization in FA-M cells, further suggesting tinatedhere.However,thefaintpresenceofFANCD2- a role for FANCM, not only in the stabilization of Lintheseextractsmightpointtoadownstreamfunc- the core complex, but also in its nuclear localization tion for FANCI, in binding of FANCD2 to the chro- [96]. matin,butalsointhestabilizationofFANCD2-L[84]. Fig.4.InFA-Jcells,bothformsofFANCD2(FANCD2-Sand-L)areclearlydetectableinnuclearextractsrepresentingthechromatinfraction. Inaddition,inFA-Icells,bothformsofFANCD2(FANCD2-Sand-L)arehardlydetectableinthechromatinfractionsofnuclearextracts.
Description:The list of books you might like
