WIXLIB

Structural insights into Non-Homologous End Joining (NHEJ)Susan P. Lees-MillerDepartment of Biochemistry and Molecular BiologyRobson DNA Science CenterArnie Charbonneau Cancer InstituteCumming School of MedicineUniversity of CalgaryAlbertaCanadaNIH DNA Repair Group April 20th 2021

Phosphatidyl inositol 3-kinase-like protein kinases PIKKsDNA-PKcsATMATRDNA-dependent protein kinase catalytic subunit (PRKDC)Ataxia-Telangiectasia MutatedATM and Rad3-relatedATMATRDNA-PKcsSerine/threonine protein kinasesActivated in response to DNA damagePhosphorylate substrates on SQ/TQ and other motifsFrom: Blackford and Jackson, Molecular Cell, 2017

Cellular response to IR-induced DNA double strand breaks (DSBs)Other DSB inducing agent Some chemotherapeutics Endogenous processesIonizing Radiation Radiation Therapy Radiation ExposureG2ResectionKu70/80MRNCtIP, Exo1,Dna2DNA-PKcsATRATMATRIPNon-homologousEnd Joining(NHEJ)DDR signalingCell cycle ation forksS phaseYXLF, XRCC4,DNA ligase 4ArtemisRPA

1985- Human cells contain a DNA-activated protein kinaseWalker et al, EMBO J 4(1) 139-145 (1985)- DNA- Hsp90Carl W. Anderson Ph.DBrookhaven NationalLaboratory, NY

1989Identification of DNA-PK phosphorylation sites inHsp90PEETQTQFirst example of SQ/TQ motif for PIKK1990Preliminary purification and characterization ofDNA-PKcs (p350) and co-purification with Ku

Simultaneous discovery of DNA-PKcsTim Carter, William Dynan, Steve Jackson

The Phosphatidyl Inositol 3-kinase-like family of serine/threonine protein kinases, PIKKs1995

Carl W. Anderson Ph.DBAPhDPDF19752011Harvard 1966Washington University 1970Cold Spring Harbour 1971-1974Biology Department, Brookhaven National Laboratory, NYChair, 1999-2011Senior Scientist EmeritusVisiting scientist at National Institute of Environmental HealthSciences (NIEHS), NCResearch interests: Poliovirus genome Discovery of DNA-PK, early work on DNA-PK purification,phosphorylation, and cloning. Post-translational modification of p53 with Ettore Appella NIH. Founded the International Association for Protein Structure Analysisand ProteomicsOctober 21 2020Age 76

Mechanism of Non-Homologous End Joining (NHEJ): Major pathway for repair of IRinduced DSBs in human cells Required for V(D)J recombination inadaptive immune systemG2Resection Active in G0, G1, G2Ku70/80 Error proneXLF, XRCC4,DNA ligase 4ArtemisMRNCtIP, Exo1,Dna2DNA-PKcsATRATMATRIPNon-homologousEnd Joining (NHEJ)DDR signalingCell cycle ation forksS phaseY Rapid repairRPA

DNA-PKcs and Ku70/80 assemble on ends of dsDNA to form DNA-PK Ku binds with high affinity to ends ofdsDNA In the presence of DNA-PKcs, Kutranslocates inwards so that DNAPKcs now occupies the extreme endsof the DNA (Yoo and Dynan, NAR, 1999) DNA-PKcs plus Ku assembled ondsDNA DNA-PKKu70/80Ku70/80Ku70/80DNA-PKcsActiveprotein kinase

Autophosphorylation of DNA-PK results in disruption of the Ku-DNA-PKcscomplex and loss of activityPPNPAMATP p-DNA-PKcsPre-incubation with ATPresults in loss of DNA-PKactivity Autophosphorylation of purifiedDNA-PKcs, Ku70 and Ku80Autophosphorylationdisrupts the interaction ofDNA-PKcs and Ku AMP-PNP- ATPp-Ku80p-Ku70 ATPPurified: DNA-PKcs, Ku70/80IP:Ku70/80WB:DNA-PKcs and KuChan and Lees-Miller, JBC, 1996

Identification of DNA-PKcs autophosphorylation sites1892N-HEATABCDE and S3205T26092056 and PQRT3950PQRS2056PM-HEATDouglas et al, BJ, 2002Chan et al, G&D, 2002Cui et al MCB 2005; Chen et al, JBC, 2005Douglas et al, MCB, TT3950PKIN35804100FATC4128

Autophosphorylation of ABCDE and PQR phosphorylation sites are important for DSBrepair and VDJ 00FATC4128 DNA-PKcs- DNA-PKcsDNA-PKcs null cells re-expressing DNAPKcs-ABCDE A are more sensitive to IRthan cells lacking DNA-PKcs- ABCDE AKathy MeekMichigan State UniversityDing et al, MCB 2003

Model for assembly of Ku and DNA-PKcson dsDNA endsKu70/80 Ku binds DSBKu70/80DNA-PKcs Recruits DNA-PKcs Assembly of a synaptic complex on bothends of the DSB (active DNA-PK) Autophosphorylation of DNA-PKcs (intrans?) Dissociation of csKu70/80PPDNAPKcsPKu70/80

Ablation of ABCDE sites impairs release of DNA-PKcs from DNA-bound Ku in vitrominPurified DNA-PKcs and KudsDNA-Biotin streptavidin-bead pull down assaysWestern blot DNA-PKcs and KuHammel et al, J. Biol. Chem. 2010Jette et al, Prog. Biophys. Mol. Biol. 2015

In vivo evidence for autophosphorylation induced dissociation of DNA-PKcs from DSBsUV laser microbeam irradiation - GFP-DNA-PKcsWt-DNA-PKcs rapidly dissociates fromUV laser induced DNA damagePhosphorylation defective(ABCDE: T to A)and kinase deadare retainedUematsu et al, J Cell Biol. 2007 (David Chen)

Model for assembly and autophosphorylation induce disassembly of the DNA-PK complexin the initial stages of NHEJ:P KuP DNA-PKcsPPDissociation ofphosphorylatedDNA-PKcsP DNA-PKSynapsis(tethering of DNAends nal change

Model for NHEJ incorporating detection of DSB by Ku, recruitment and autophosphorylation ofDNA-PKcs, removal of non-ligatable end-groups and ligation1. IR induces DSBs withnon-ligatible end groups Ku* *2. Detection of DSB3. Tethering of DSB ends Autophosphorylation ofDNA-PKcs Processing (removal ofnon-ligatable end-groupsproduced by IR)1. DSB detectionand tethering:Ku, DNA-PKcs3. Ligation:XRCC4, DNAligase IV, XLF**PNKP DNA-PKcs2. End Processing:QuickTime and adecompressorare needed to see this picture.Removal of IR-inducednon-ligatable end groups *P PLigation of DSB endsP PP PP PDNA-PK Removal of sociation of autophosphorylatedDNA-PKcs

Model for NHEJ incorporating detection of DSB by Ku, recruitment and autophosphorylation ofDNA-PKcs, removal of non-ligatable end-groups and ligationOrder of events?* * Ku3. Ligation:1. DSB detectionand tethering:XRCC4, DNAligase IV, XLFKu, DNA-PKcsOrder of recruitment?Artemis interacts withDNA-PKcs and DNAligase IV. How/when isArtemis recruited?PNKP interacts withXRCC4. When is XRCC4recruited? When doesprocessing occur?**ArtemisPNKP? DNA-PKcs?2. End Processing:QuickTime and adecompressorare needed to see this picture.Artemis, PNKP, Pol mu/pol lambdaMre11? Tdp1? WRN1?P PP PP PP tion of autophosphorylatedDNA-PKcs

Assembly of a molecular machineDetection, tethering, end-processing and ligation all occur within one dynamic molecular assemblyDSB ends always enclosed within assembly, hand-off from one stage to anotherDetectionProcessingLigation and releasePP DNA-PKcs phosphorylationP ATM phosphorylationPCK2 phosphorylationAdapted from Williams et al, DNA Repair, 2014Yano and Chen, Cell Cycle 2008John TainerLBNL/MD Anderson

How does the NHEJ machinery assembleand carry out NHEJ?APLFLINP1 How does the XLF-XRCC4-DNA ligase IV complexinteract with the Ku-DSB-DNA-PKcs complex? How does Artemis interact with DNA-PKcs andDNA ligase IV?DNALigase IV How do PAXX, APLF and LINP1 stabilize thesynaptic complex?Hammel et al, JBC, 2016Brosey et al, Methods Enzymol, 2017Wang et al, Nature Struc. Mol. Biol. 2018Thapar et al, NAR 2020XLFXRCC4

Structures of NHEJ componentsStructure of the Ku70/80 heterodimer,core DNA binding domainKU80 contains a flexible C-terminal “tail” thatinteracts with DNA-PKcsRed KU70Yellow KU80Grey dsDNAWalker et al, Nature 2001Hammel et al, JBC, 2010Gell and Jackson, NAR, 1999Harris et al, J. Mol. Biol. 2004

Structures of DNA-PKcs and DNA-PKcs-Ku-dsDNA ibanda et al, Nature, 2010Sharif et al, PNAS, 2017Chen et al, Molec Cell, 2021Chaplin et al, Nat. Struc.Molec. Biol. 2021Sibanda et al, Science, 2017ABCDET2609, S2612T2620, T2638S3205T2647 2801PFAT3580T3950P 4100KINFATC4128Yin et al, Cell Reports, 2017

Structures of NHEJ componentsXRCC4Minus C terminal tailJunop et al, EMBO J, 2000Sibanda et al, NSMB, 2001Wu et al, MCB, 2009Andres et al, Mol. Cell, 2007XLF (XRCC4-like factor)Minus C terminal tailXRCC4 (truncated, blue) and XLF(truncated, magenta) interact viatheir head domainsMichal HammelHammel et al, JBC, 2011

Models for how the NHEJ proteins may interact at DSBs based on individual structuresFrom Frit et al, 2019Hammel et al, JBC, 2011, 2016Frit et al, Prog Biophys Mol Biol. 2019Liang et al, Prog Biophys Mol Biol. 2020Graham et al, Molec. Cell 2016

Challenges with understanding the function of DNA-PKcsIts BIG4128 amino acids180 x 130 x 1051892N-HEATSibanda et al, Science, PPP2801S3205PFATT3950PKIN35804100FATC4128

What’s the difference between these model organisms?Contain DNA-PKcs/PRKDCNo DNA-PKcs/PRKDC“DNA-PKcs is a vertebrate-specific PIKK”HumanDrosophila melanogasterMouseDanio rerio (Zebrafish)S. pombeArabidopsis thalianaCaenorhabditis elegansS. cerevisiae

DNA-PKcs is highly conserved in vertebrateslcl Query 10001lcl Query 10002lcl Query 10003lcl Query 10001lcl Query 10004lcl Query 10002lcl Query 10005lcl Query 10003lcl Query 10006lcl Query 10004lcl Query 10007lcl Query 10005lcl Query 10008lcl Query 10006lcl Query 10009lcl Query 10007lcl Query 10010lcl Query 10008lcl Query 10011lcl Query 10009lcl Query 10012lcl Query 10010lcl Query 10013lcl Query 10011lcl Query 10014lcl Query 10012lcl Query 10015lcl Query 10013lcl Query 10016lcl Query 10014lcl Query 10017lcl Query 10015lcl Query 10018lcl Query TORMILIIGARBAMBOOlcl Query 10017lcl Query 10018ALLIGATORGARCOBALTLees-Miller et al, Prog. Biophys. Mol. Biol. 2020Red highly conservedBlue less conservedGrey no conservation

DNA-PKcs is highly conserved from human to sponge(metazoa/vertebrates/invertebrates)COBALTRed highly conservedBlue less conservedGrey no conservationLanceletAnnelid wormScorpionPolychaeteAlvinella pompejanaSea urchinTrichoplaxPlacazoaCoralLees-Miller et al, Prog. Biophys. Mol. Biol. 2020***

Weak amino acid similarity between DNA-PKcs and other PIKKsAmino acid identitybetween DNA-PKcsATM, ATR and mTORFrom Clustal OmegaDNA-PKcsHuman: 100%Mouse: 80%Xenopus 64%Danio58%ATMATRmTOR 18% 17% 16%COBALTRed highly conservedBlue less conservedGrey no conservation

DNA-PKcs conservation in representatives from all major phylaCOBALT Red highly conservedBlue less conservedGrey no conservationRotiferBrachionus plicatilisPlantMossPhyscomitrella patensNematodeRoundwormTrichinella pseudospiralisOomyceteLeaf moldPlasmopara halstediiLees-Miller et al, Prog. Biophys. Mol. Biol. 2020***

With a few exceptions, DNA-PKcs/PRKDC is present in all major eukaryotic phyla including molds, non-floweringplants, algae, amoeba and invertebrates.Nematodes:EnopleaNOT in ChromadoreaLepidoptera ?FungiChytridomycotaMycomycotaNOT in DikaryaNot infloweringplantsGreen AlgaeClub mossPine, spruce, fern,Cycads, bBugsDNA-PKcs is NOT a vertebrate-specific PIKKHorsehoe vogeneao.s3.amazonaws.com/images/tree of life/tree-of-life 2000.png

Highly conserved regions within DNA-PKcsForehead892-1289Lees-Miller et al, Prog. Biophys. Mol. Biol. 2020Kinase domain3580-4099FATC4100-4128

Conservation of the YRxGxxPD motif in DNA-PKcs from humans to molds1PQR892M-HEATN-HEATForehead892-1289PABCDE 2801Nuc1815-2202 ABCDEPFATPKIN35804100FATC4128YRPDLoop2577-2773 nephthya giganteaSpongeAmphimedon queenslandicaStarfishAcanthaster omum lignanoPlantsGinko myceteLeaf moldPlasmopara halstediiGinko biloba

YRPD motif is located between ABCDE loop and FAT 1815-2202ABCDET2609, S2612T2620, T2638S3205T2647 2801PFAT3580T3950P mel et al, Prog. Biophys Mol. Biol. 2020; Tsutakawa in Lees-Miller et al, Prog. Biophys Mol. Biol. 2020FATC4128

APLFLINP1Assembly of the NHEJ machine?DNALigase IVXLFHammel et al, JBC, 2016Brosey et al, Methods Enzymol, 2017Wang et al, Nature Struc. Mol. Biol. 2018Thapar et al, NAR 2020XRCC4

Assembly of DNA-PKcs-Ku-DNA on dsDNA in a synaptic complexHydrogen/Deuterium Exchange Mass SpectrometryCross-linking mass spectrometryHepburn et al, Structure, 2020Dave SchriemerUniversity of Calgary

oDNA-PKcs-Ku-DNA synaptic complex at 13 AoDSB ends offset by 115ALong-Range Synaptic complex: Graham et al, 2016DNA-PKcs residues 2577-2773 (disordered loop containing ABCDE phosphorylation sites)form “plug” blocking the passage of dsDNAHepburn et al, Structure, 2020