Repair, Reuse, Recycle-The Expanding Role of Autophagy in Genome Maintenance.pdf

ReviewRepair,Reuse,Recycle:TheExpanding Role of Autophagyin Genome MaintenanceGraeme Hewitt1,*and Viktor I.Korolchuk2,*(Macro)Autophagy is a catabolic pathway that delivers excess,aggregated,ordamaged proteins and organelles to lysosomes for degradation.Autophagy isactivated in response to numerous cellular stressors such as increased levels ofreactiveoxygen species(ROS)andlow levelsofcellular nutrientsas wellasDNAdamage.Although autophagy occurs in the cytoplasm,its inhibition leads toaccumulation of DNA damage and genomic instability.In the past few years,ourunderstanding of the interplay between autophagy and genomic stability hasgreatly increased.In this review we summarize these recent advances in under-standing the molecular mechanisms linking autophagy to DNA repair.IntroductionMaintenance of genomic integrity is essential for organismal survival.DNA can be damaged by aplethoraofextrinsicfactorssuchasUVradiation,ionizingradiation,andchemicalcompoundsaswell as intrinsic factors such as free radicals generated as part of normal metabolism andmistakes in replication.This barrage ofgenotoxic insults resultsin thegeneration of an estimated19 200 DNA lesions per day 1.It is therefore necessary for cells to have effective mechanismsfor sensing and repairing this damage to maintain their survival.Like genomic integrity,maintenance of protein homeostasis is an essential requirement for celland organismal survival.Thus,there are multiple pathways responsible for the turnover ofdamagedandunwantedproteinsandorganelles.Theubiquitin(Ub)proteasomesystem(UPS)isresponsible for the degradation of short-lived,soluble proteins,is active in both the cytoplasmand the nucleus,andis thoughtto account for themajority ofprotein turnover in the cell.Taggingof target proteins with Ub chains makes theUPS highly selective.As a result of its ability to tightlycontrol proteinlevels,theUPShasbeen shownto regulatenumerous cellular processessuchasthe cell cycle,signal transduction,and DNA repair 2.The term autophagy translates from the Greek auto meaning oneself and phagy meaning toeat and describes evolutionarily conserved catabolic cellular degradation pathways involved inthe delivery of cytoplasmic cargo to the lysosome.There are three main types of autophagy ineukaryotic cells:microautophagy(direct engulfment and degradation of portions of the cyto-plasm via invagination of thelysosome 3);chaperone-mediated autophagy(direct translocationof targeted proteins containing the KFERQ motif into the lysosome via the LAMP-2A receptor4);and macroautophagy(sequestration of cytoplasmic cargo in double-membrane vesiclescalled autophagosomes followed by transport along microtubules and fusion with the lyso-some).Macroautophagyisthefocusofthisreviewandishereinreferredtoasautophagy.Similarto the UPS,cargo can be targeted for autophagy by the selective action of dedicated receptorproteins.Also as in the UPS,the main signal for autophagic degradation is the ubiquitination ofcargo allowing its recruitment to the autophagosome by the receptors.Given the absence ofTrendsRecent studies have demonstrated theturnover of nuclear components suchas nuclear lamina,chromatin,and DNAby autophagy and suggest that it playsan important role in maintaining geno-mic stability.Loss/inhibition of autophagy gives riseto reduced DNA damage repair andincreased cell death in response togenotoxic stress.The accumulation of the autophagyreceptor protein p62/SQSTM1 thatresults from inhibition/loss of autop-hagy leads to inhibition of double-strand break(DSB)repair throughhomologous recombination(HR).Recently,progress has been made inunraveling the molecular mechanismslinking p62 and DSB repair.Nuclearp62 dampens HR through the inhibi-tion of RNF168-mediated chromatinubiquitination as well as targeting theHR proteins RAD51 and filamin A fordegradation via the proteasome.1DSB Repair Metabolism Laboratory,The Francis Crick Institute,LondonNW1 1AT,UK2Institute for Cell and MolecularBiosciences,Newcastle University,Newcastle upon Tyne NE4 5PL,UK*Correspondence:graeme.hewittcrick.ac.uk(G.Hewitt)and viktor.korolchukncl.ac.uk(V.I.Korolchuk).340Trends in Cell Biology,May 2017,Vol.27,No.5http:/dx.doi.org/10.1016/j.tcb.2016.11.011 2016 The Authors.Published by Elsevier Ltd.This is an open access article under the CC BY license(http:/creativecommons.org/licenses/by/4.0/).autophagy in the nucleus and the previous belief that autophagy was a nonselective process,itwas a long-held belief that the UPS was the sole degradation pathway impacting DNA damagerepair.Recently,however,evidencehasbeenaccumulatingtosuggestthatautophagyandDNAdamage repair may be mechanistically linked despite occurring in spatially distinct cellularcompartments.In this review we provide an overview of the current model of the core machineryof autophagy and the DNA damage response(DDR).We then focus on recent developments inour understanding of the role of autophagy in genome maintenance and DNA repair.AutophagyAutophagy plays a central role in the maintenance of cellular homeostasis and is active at basal,albeit varying,levels in all cells.Basal autophagic activity is dependent on cellular function as wellas energy demand.Autophagy serves to dispose of damaged or unwanted proteins andorganelles that accumulate as a consequence of normal cellular metabolism,like toxic proteinaggregates and damaged mitochondria(reviewed in 5).Its activity is governed by numeroussensorsthatdetectchangestotheintra-andextracellularenvironment,meaningthatautophagycan be activated in response to various cell stresses such as starvation,hypoxia,mitochondrialdamage,and pathogen infection.The primary purpose of this activation is to maintain cellularhomeostasis and promote cell survival and disruption of the autophagic process has beenimplicated in the pathology of many human diseases,from cancer to age-related neurodegen-eration 6.Excessive activation of autophagy has also been observed in cells undergoing cell death.Theexact role of this activation remains an area of debate as it is unclear whether cells die as a directconsequence of autophagic activation or whether autophagy is active in fatally stressed cellsmerely as a last-ditch,albeit failed,attempt to promote survival(reviewed in 7).Recently,it hasbeen suggested that autophagy may playa causative role in cell death in a pathway distinct fromapoptosis or necroptosis 8.Here,excessive activation of autophagy through treatment withautophagy-inducing peptides,starvation,or cerebral hypoxiaischemia was shown to inducecell death via a mechanism dependent on cellular Na+,K+-ATPase.The prosurvival and possiblepro-cell-death roles place autophagy at the center of the maintenance of cell and organismalhemostasis in response to various stresses.During the process of autophagy,lipid membranes,the origin of which is debated,assemble inthe cytosol giving rise to a nascent autophagic vesicle(the autophagosome)that engulfs cargodestined for degradation.Once the autophagosome is sealed,it is transported along micro-tubules and eventually delivers its cargo to the lysosome for degradation by forming a hybridstructure termed the autolysosome(Box 1).This process not only allows the removal ofdamaged,potentially toxic cellular components but also enables the release of basic molecules(e.g.,amino acids,lipids)back to the cytoplasm for reuse in biosynthetic or catabolic processesas building blocks and a source of energy 9.Initially believed to be a nonselective process,autophagy is now known to show specificitytowards various intracellular substrates.This specificity is mediated by autophagic receptorssuch as the prototypical member of the family p62/SQSTM1 as well as several functionallyrelated proteins 10.Most of the receptor proteins contain Ub-binding domains,allowing themto interact with cargo tagged for autophagic degradation with Ub chains including Lys63poly-Ub and other types of linkage 11.While not considered to be required for autophagosomalbiogenesis itself,receptor proteins mediate the docking of autophagic membranes to cargo andpromote its sequestration by the nascent autophagosome.This function is mediated by LC3-II-interacting region(LIR)motifs 12,which allow interaction with autophagy(Atg)8-family proteinsontheinnersurfaceoftheautophagosome(Box1).Receptor-mediatedselectiveautophagycantarget various subcellular components and can be classified intosubtypes including aggrephagyTrends in Cell Biology,May 2017,Vol.27,No.5341(clearance of aggregate-prone proteins),mitophagy(dysfunctional mitochondria),and nucle-ophagy(damaged nuclear components)13.Double-Strand Break(DSB)RepairDSBs are highly toxic lesions where both strands of DNA are broken.They have been shown tocause genomic instability and cell death.To protect against this,organisms have evolved highlyregulated DDR mechanisms for sensing of the damage(Box 2)and activation of DSB repairBox 1.Autophagic MachineryThe process of autophagosome formation is carefully orchestrated by over 30 core Atg proteins and a plethora ofassociated regulatory components.The initiation step in the formation of the nascent autophagic vesicle(also called aphagophore or an isolation membrane)requires the activity of the Atg1(or ULK1)complex(Figure I).The protein kinaseULK1 is activated in response to nutrient deprivation,which is mediated by the suppression of an upstream proteinkinase,mTOR complex 1(mTORC1).Inhibition of mTORC1 relives ULK1 from its inhibitory phosphorylation allowing it toform a complex with the Atg13,FIP200,and Atg101 proteins 68.The nucleation event,which follows initiation,is drivenby a second key protein complex involving a phosphatidylinositol 3-kinase(PI3K),Vps34.Together with Vps15/p150,Vps14,and Vps30/Atg6(Beclin-1 in mammals),Vps34 produces phosphatidylinositol 3-phosphate(PI3-P),a lipid thatstarts the nucleation event in the formation of the autophagosome 69.Phagophore elongation,a crucial step in autophagosome formation,involves the action of two Ub-like conjugationsystems.The first promotes the conjugation of the phosphatidylethanolamine(PE)molecule to the C terminus of Atg8-family proteins(Atg8/LC3-II/GABARAP).These small Ub-like proteins are synthesized as precursors that are cleaved attheir C termini by the cysteine protease Atg4 70.The cleaved forms of these proteins(e.g.,in the case of LC3,calledLC3-I)are subsequently conjugated to PE by the consecutive actions of the Atg7 and Atg3 enzymes,thus producing thelipidated form(e.g.,LC3-II).The lipidated form of Atg8 proteins is specifically associated with,and is required for theelongation of,the autophagosomal membrane 71.In the parallel conjugation reaction,the Atg7 and Atg10 enzymesmediate covalent attachment of Atg12 to Atg5 72.The Atg5Atg12 conjugate in a complex with Atg16 assists in thelipidation of Atg8 proteins and the extension of the phagophore 73,74.Following the completion of the autophagicvesicle,its maturation involves fusion with an endosome(thus forming the amphisome)or directly with a lysosome,ultimately leading to the formation of the degradative organelle,the autolysosome.Figure I.The Core Machinery of(Macro)Autophagy.342Trends in Cell Biology,May 2017,Vol.27,No.5pathways(Figure1).Defectsintheseprocesseshavebeenshowntolead tonumerousdiseasesas well as to drive cancer development 14.There are two main pathways responsible for the repair of DSBs:non-homologous end joining(NHEJ)and homologous recombination(HR)(Figure 1).NHEJ is active throughout the whole ofthe cell cycle whereas HR is active only during the S and G2phases when sister chromatids areavailable as templates for repair.The use of a homologous template by HR allows error-freerepair 1523.NHEJ,by contrast,relies on the ligation of DNA ends without the use of atemplate,resulting in an increased occurrence of erroneous repair products 24.Inhibition orabsence of HR leads to reliance on the error-prone NHEJ pathway,leading to increasedmutations and chromosomal rearrangements.Key components of the HR pathway suchBRCA2 and RAD51 are known tumor suppressors,with inactivating mutations being linkedto genomic instability 25,26.Besides DSB repair,HR factors are important for the protection ofstalled replication forks in a recombination-independent manner 27.RAD51 is recruited tostalled replication forks through an Mre11-and BRCA2-dependent mechanism.In a tightlybalanced process,Mre11 is thought to extend the single-stranded DNA(ssDNA)gap left by thereplisome and to facilitate post-replication repair while BRCA2 leads to RAD51 loading,whichprotects arrested forks by preventing overextension of ssDNA gaps 27.By contrast,NHEJ does not require a homologous template to mediate DSB repair.Instead,DNA ends are directly joined by DNA ligases(Figure 1)24,2833.NHEJ is not only required torepairDSBsgeneratedbyexogenousandendogenousstress;itisalsoneededfortheligation ofbreaks generated during V(D)J recombination,a process required for immunoglobulin and T cellreceptor(TCR)diversity 34.Immunoglobulin and TCR genes contain variable(V),diversity(D),and joining(J)sections that are joined by the NHEJ machinery giving rise to mature V(D)J exonsinBandTcells,respectively35.DSBsaregeneratedbetweenrecombinationsignalsequences(RSSs)and coding DNA by the RAG1 and RAG2 proteins 36.The resulting DSBs have bluntBox 2.The DDRDSBs,as well as ssDNA,lead to potent activation of the DDR(Figure I).Specialized sensing complexes are responsibleforthedetectionofDNAlesionsandinitiationoftheDDR.Initially,DSBsaresensedandprocessed bytheMre11complex(MRN)comprisingf Mre11/Rad50/Nbs1,while ataxia telangiectasia and Rad3 related(ATR),ATR-interacting protein(ATRIP),replication protein A(RPA),Rad9/Rad1/Hus1,and Rad17/RSR are all involved in sensing ssDNA.Followingdetection,sensors recruit phosphoinositide kinase-related kinases(PIKKs),ATR,and ataxia telangiectasia mutated(ATM)to the site of damage leading to the local phosphorylation of histone H2A.X(H2A.X)to gH2A.X 75.AtDSBsthe ATM-mediatedphosphorylationofH2A.XleadstofurtherrecruitmentofATM,setting upapositivefeedbackloop that results in the spread of gH2A.X up to 2 Mb from the site of damage 76.This amplification step is furtherfacilitated by the DDR mediators mediator of DNA damage checkpoint 1(MDC1)and p53-binding protein1(53BP1)77and establishes a platform to which other DNA repair factors are recruited.The recruitment of 53BP1 and BRCA1 playsan important role in DNA repair pathway choice.BRCA1 has been shown to promote the removal of 53BP1 during Sphase,promoting DNA resection and repair through the HR pathway 78.Conversely,53BP1 has been shown tonegatively regulate resection in G179.At SSBs stranded DNA is bound by RPA,signaling the recruitment of ATR 80.ATR activity is further amplified by theheterotrimeric 911 complex(RAD9,RAD1,and HUS1)and topoisomerase-II-binding protein 1(TOPBP1)81,82.Downstream of TOPBP Claspin leads to the activation of CHK1 83.Both CHK1 and a related protein kinase,CHK2,arephosphor