WELCOME TO THE DNA REPAIR INTEREST GROUP VIDEO CONFERENCE SERIES. IF YOU WANT TO GET ON THE MAILING LIST, YOU CAN JUST SEND AN E-MAIL TO NIH.GOV AND YOU WILL BE NOTIFIED OF THESE VIDEO CONFERENCES, WE'VE BEEN GOING ON THIS FOR DECADES AND THERE ARE MANY THAT ARE ARCHIVED NOW. WE HAVE ONCE A MONTH IN MAY 18th, FROM NIEHS, WE'LL TAKE ABOUT MUTATIONAL SIGNATURES OF STRESS IN YEAST IN MEN AND AND A DOCTOR FROM THE LAB WILL BE SPEAKING ON SHORT TERM NAD AND SUPPLEMENTATION PRESENTS HEARING LOSS IN MASS MODELS OF SYNDROME AND WE HAVE ANOTHER ONE SCHEDULED FOR WE HAVE DR. SEUSS AN LEES-MILLER HERE. >> IT'S A PLEASURE TO HAVE YOU HERE TO TALK ABOUT YOUR LATEST VERY EXCITING RESULTS AND JOINING SUSAN IS WELL-KNOWN TO ALL OF US IN DNA REPAIR AND JOINING WHERE SHE'S BEEN A TRUE LEADER FOR MANY YEARS AND ALWAYS A VERY GENEROUS COLLABORATE ERR ON HELPFUL WITH ADVICE, YOU'VE GIVEN ME ADVICE, GIVEN ME REAGENTS THAT WORKED BEAUTIFULLY SO THAT'S MUCH APPRECIATED. SUSAN IS FROM WALES, I HOPE WE CAN UNDERSTAND EVERYTHING YOU SAY. [LAUGHTER] AND SHE DID HER PHD IN WALES AND SHE MOVED TO CANADA AND HAS BEEN IN ALBERTA. SHE DID A POST DOCK FELLOWSHIP THERE IN ALBERTA AND BECAME SENIOR RESEARCH ASSOCIATE AND LATER ON A SCIENTIST AND LATER ON SHE BECAME AN ASSISTANT PROFESSOR THERE AND BECAME A FULL PROFESSOR AND HAS ALSO HAD ADDITIONAL APPOINTMENTS TO THE DEPARTMENT OF ONCOLOGY, AND DEPARTMENT OF BIOCHEMISTRY IN ALBERTA. YOU HAVE MANY MEMBERSHIPS OF ASSOCIATIONS AND IMPORTANT THINGS AND HAVE MANY ACHIEVEMENTS, YOU ARE ALSO A LEADER OF THE DROP DNA SCIENCE CENTRE IN CALGARY AND ON THE GENOME AND STABILITY AND AGING GROUP THERE. SO WE'RE VERY PLEASED THAT YOU WOULD COME AND VISIT US HERE ON THE VIDEO AND WE LOOK FORWARD TO YOUR TALK. THANK YOU. >> I HAVE A COUPLE MORE WORDS TO ADD, AS YOU WILL BE HEARING, SHE DID DO A POST-DOC AT BROOKE HAVEN LAB AND YOU WILL HEAR ABOUT THAT AND I WAS GOING THROUGH HER VERY EXTENSIVE C.V. WITH WONDERFUL, HIGHLY CITED PAPERS AND IN THE MIDDLE OF IT WE HAVE NOTION THAT WE HAVE NOT SEEN IN THE UNITED STATES, IT'S CALLED "CAREER INTERRUPTIONS" DURING THE TIME SHE HAD MATERNITY LEAVE FOR HER THROW NOW-GROWN CHILDREN. OVER ALL, THAT WAS THERE BEFORE. IT'S A VERY HUMANE ADDITION TO C.V.s AND IN THE UNITED STATES WE MIGHT THINK ABOUT THAT. THE TALK TODAY IS STRUCTURAL INSIGHTS INTO NON HEMMOLOGIST. >> THANK YOU SO MUCH, KEN, FOR THE INVITATION TO PRESENT TO THE GROUP TODAY. IT'S A REAL HONOUR. THANK YOU. I WORK IN DNA DOUBLE STRAND BREAK REPAIR AND THE ROLE OF THE PHOSPHATIDYL AND MUCH OF MY WORK HAS BEEN DONE ON DNA-PK-CS SO DNA PIKKs PLAY IMPORTANT ROLES IN THE CELLULAR RESPONSE TO DNA DOUBLE STRAND BREAKS AND DNA-PK IN PARTICULAR, INTERACTS WITH THE PROTEIN AND IS RECRUITED TO DNA DOUBLE STRAND BREAKS AND THIS IS THE INITIATING STEP IN THE PATHWAY SO I'M SURE MANY OF YOU SEE IN THIS SLIDE A VERY FAMILIAR WITH THE DIFFERENT PROTEINS AND THE DIFFERENT PATHWAYS ON HERE, BUT WHAT YOU MIGHT NOT KNOW IS THAT THIS WHOLE FIELD AND THE DISCOVERY OF DNA-PK-CA BEGAN WITH CARL ANDERSON, A FACULTY MEMBER AT BROOKE HAVEN NATIONAL LAB OR LABORATORY IN NEW YORK. CARL HAD MANY DIFFERENT INTERESTS IN POST TRANSLATION AND TRANSLATION AND HE WENT TO DO A SABBATICAL IN THE U.K. TO WORK ON THE RNA DEPENDENT PROTEIN SO THEY PURCHASED TRANSFER FROM SIGMA AND AND THEY GOT UNEXPECTED RESULTS WHICH THEY FOLLOWED UP AND IT TURNED OUT THE RNA THEY WERE USING WAS CONTAMINATED WITH DOUBLE STRANDED DNA AND THAT'S ACTUALLY WHAT LED TO THE DISCOVERY OF THIS DNA-ACTIVATED PROTEIN KEEN ACE AND THIS IS A PICTURE FROM THAT FIRST PAPER AND YOU CAN SEE DIFFERENT PROTEINS AND ONE THAT CARL IDENTIFIED WAS HSP-90. I WAS VERY FORTUNATE TO GO TO CARL'S LAB TO DO MY POST-DOC BECAUSE I HAD A PACK GROUND AND INTEREST IN PROTEIN FOSS SO WE PURIFIED AND CHARACTERIZED THIS DNA ACTIVATED KINASE AND WE SHOWED IT FOSS FOLLOWATED HSP90 ON THIS PRO TVUSD WHICH WAS A FIRST EXAMPLE OF AN SQTQ MOTIF WHICH IS THE SIGNIFICANT FOR THE PIKK FAMILY OF PROTEINS. SO, WE WEREN'T THE ONLY ONES FOLLOWING UP ON CARL'S INITIAL DISCOVERY. TIM CARTER ALSO PURIFIED A DNA ACTIVATED PROTEIN KINASE AS DID BILL DIAMOND AND HE HAD LOVELY WORK IN THE EARLY DAYS OF DISCOVERY OF DNA-PK AND STEVE JACKSON IN CAMBRIDGE, WHO HAS MADE MAJOR CONTRIBUTIONS TO THE FIELD. AND CARL COLLABORATED WITH STEVE ON THE CLONING OF DNA-PK CS AND IN 1995 WE SHOWED THE DNA-PK CS WAS RELATED TO A.T.M. AND A MEMBER OF THE NEWLY DISCOVERED PIKKs PROTEIN OF KINASES. I JUST WANTED TO MENTION THESE PAPERS BECAUSE SADLY, CARL PASSED AWAY IN OCTOBER OF LAST YEAR AFTER A BATTLE WITH CANCER AND HE WAS A VERY MODEST MAN AND A GREAT MENTOR AND COLLEAGUE AND FRIEND. I THINK HE MADE A NUMBER OF REALLY IMPORTANT CONTRIBUTIONS, NOT ONLY TO THE DNA-PK AND THE PIKK FIELD BUT TO P-53 AND HE HAD A VERY LONGSTANDING COLLABORATION AT THE NIH ON P-53. SO, AS I SAID, WHAT I'M GOING TO TALK ABOUT TODAY IS IS THE MAJOR PATHWAY FOR THE REPAIR OF RADIATION INDUCED DNA DOUBLE STRAND BREAKS IN HUMAN HELLS AND IT'S REQUIRED FOR VDJ RECOMBINATION AND THE IMMUNE SYSTEM AND IT'S ACTIVE AND CARRIED OUT REPAIR THOUGHT TO BE ERROR PRONE. GOING RIGHT BACK TO THE BASICS, THE INITIATE AND STEPS IN NON-HOME OLE GUS ARE RECRUITMENT OF THE K PROTEIN AND IT'S BOUND TO THE END 69 DOUBLE STRAND BREAK AND THIS WAS SHOWN MANY YEARS AGO ACTUALLY FROM BILL'S GROUP. AND WHEN KU AND DNA-PK ARE ASSEMBLED ON AN END OF DOUBLE STRANDED DNA IT FORMS THE ACTIVE PROTEIN KINASE. AND SO, FOR MANY YEARS, WE'VE KNOWN THAT DNA-PK CS UNDERGOES AUTO FOSS FOLLOWATION SO IT SHOWS SHOWN WHEN I STARTED MY LAB AND YOU SEE IT LOOSES ACTIVITY IN YOU ARE IN THE ABSENCE OF ATP OR THE ANALOG A M.P.P. M.P. YOU CAN SEE THAT OF A DECEMBER IS RETAINED AND SO IT UNDER GOES AS OWE PHOSPHORYLATION AND WHEN IT PHOSPHORYLATION IT LOSES ITS ACTIVITY AND WE FOUND YOU COULD THE KU COMPLEX IN THE ABSENCE OF ATP OR IN THE PRESENCE OF THE NON HYDRO LIES ABLE ANALOG ANNAMP BUT IF YOU PRE INCUBATE IT WITH ATB TO REDUCE AS OWE PHOSPHORYLATION YOU CAN HAVE KU BUT YOU POST DNA-PK CS SO IT LED TO THE IDEA THAT DNA-PK CS ASSEMBLED ON THE AND WAS RELEASED AND SO WE WERE VERY INTERESTED IN FOLLOWING UP ON THE AUTO PHOSPHORYLATION AND WE AND OTHERS MAPPED ON DNA-PK CS AND THE ONES I'LL TALK TO TODAY AT THE ABCDE PHOSPHORYLATION CLUSTER, AND THIS IS A CLUSTER OF A FIVE OR SIX PHOSPHORYLATION SITES BETWEEN RESIDUES 2,600 AND 2650 AND IT'S LOCATED IN THIS PART OF THE PROTEIN IN THE HEAT REGION AND CLOSE TO THE FACT AND I'LL COME BACK TO SORT AND WE WERE VERY FORTUNATE TO HAVE A COLLABORATION WITH KATHY MEEK AND SHE DID SOME BEAUTIFUL AND CONTINUES TO DO SOME BEAUTIFUL MOLECULAR BIOLOGY WHERE SHE CAN KNOCK OUT DNA-PK CS AND REEXPRESS THE MUTE ANTS AND LOOK AT THE AFFECTS ON NON A HOMOLOGOUS AND SO THIS A FIGURE FROM ONE OF KATHY'S PAPERS FROM MANY YEARS AGO NOW AND SHOWING THAT IF YOU LOOK AT DNA-PKCS CELLS THEY'RE RADIO SENSITIVE AND IF YOU PUT BACK IN DNA-PK CS WHERE THE ABCDE SITES ARE MUTATED, THEY'RE MORE RADIO SENSITIVE THAN CELLS LACKING DNA-PK CS ALL TOGETHER. WHAT IT A -- NORMALLY THE AUTO PHOSPHATE AND THE DNA WOULD BE RELEASED, IT REMAINS STUCK TO THE DNA DOUBLE STRAND BREAK AND WILL BLOCK ANY OTHER SO THERE'S EVIDENCE FROM THIS IDEA BY CHEMICAL STUDIES THAT WE CARRIED OUT AND THESE ARE WITH PURIFIED PROTEINS WHERE WE'VE ASSEMBLED DNA-PK CS AND KU ON DNA AND DNA PULLED DOWN AS SAYS AND YOU CAN SEE IN THE ABSENCE OF ATP WE CAN BRING DOWN KU AND DNA-PK CS IF WE ADD ATP TO THE MIXTURE WE STILL BRING DOWN KU BUT WE LOSE DNA-PK CS AND WE CAN BRING DOWN THE WHOLE COMPLEX AND WHEN WE FUR PIE THE ABCDE YOU CAN SEE IT INTERACTS WITH K ON DOUBLE STRANDED BUT IT'S LESS ABLE TO DISASSOCIATE FROM KU IN THE PRESENCE OF ATP SO THIS SUGGESTS THEN THAT ABLATION OF THESE ABCDE SITES IMPAIRS THE RELEASE OF DNA-PK CS FROM DNA BOUND KU AND THERE'S SOME VERY BEAUTIFUL DATA FROM DAVID CHAN'S LAB THAT SUPPORTS THIS IDEA IN VIV HO AND THIS IS IS USING GPF LABEL IN DNA-PK RODENT CELLS AND UV LASER MICRO BEAM RADIATION AND YOU CAN SEE BY THIS RED LINE, DNA-PK CS WILD TYPE GOES TO THE BREAK VERY RAPIDLY AND THEN IS RELEASED OVER THIS TWO-HOUR TIMEFRAME WHERE AS IF YOU HAVE DNA-PK CS WITH MUTATION OF THE ABCDE PHOSPHORYLATION SITES IT GOES TO THE BREAK AND IS RETAINED FOR MUCH LONGER AND SIMILARLY, IF YOU HAVE DNA-PK CS IT'S KINASE DEAD, IT GOES TO THE BREAK AND IS RETAINED. SO, ALL OF THIS SUPPORTS THIS IDEA THAT DNA-PK CS IS RECRUITED TO THE DOUBLE STRAND BREAK, IT FORMS THIS DNA-PK COMPLEX AND WE PROPOSE THAT DNA-PK AND KU TETHER THE ENDS OF THE DOUBLE STRAND BREAK TOGETHER AND IT UNDERGOES AUTO PHOSPHORYLATION AND WE PROPOSE THIS OCCURS IN TRANS AND THIS LEADS TO DIS ASSOCIATION OF THE DNA-PK CS SO HOW DOES THIS FIT INTO NON A MOL A GUSEN DIMING, I'VE HAVING TROUBLE ADVANCING MY SLIDES. WHAT WE PROPOSED IS THAT NON HOMOLOGOUS IS IN THREE SORT OF PHASES AND THE INITIAL PHASE THAT I'VE TALKED ABOUT WHERE KU BINDS TO THE DOUBLE STRAND BREAK, RECRUITS DNA-PK CS AND THEN DNA-PK HOLDS THESE ENDS TOGETHER IN THIS SYNAPTIC COMPLEX. WE KNOW THAT RADIATION INDUCES MANY TYPES OF EP GROUPS AND SO I REPRESENTED THESE HERE BY THE LITTLE RED STARS AND SO THESE GROUPS HAVE TO REMOVED BEFORE THE DNA ENDS CAN BE LIE GATED SO WE THINK THERE'S A PROCESSING STEP AND FINALLY, THE DNA ENDS WILL BE LIE GATED TOGETHER. IT UNDERGOES THIS AUTO PHOSPHORYLATION AND HANDS OVER TO THE PROCESS AND ENZYMES AND TO THE LIBATIO LIGATION COMPLEX AND THIS MODEL BECAME MORE AND MORE PROBLEMS WITH THIS MODEL AS MORE AND MORE COMPONENTS OF THE PATHWAY WERE IDENTIFIED AND THE ROLES AND A LOOSE DATED, FOR EXAMPLE, AUTO MUSS IS A KNEW CREATES THAT INTERACTS WITH DNA-PK PC AND IN A LIE GAYS FULL AND IT INTERACTS WITH XOCC4 SO THIS IDEA OF WHAT DO THE PROTEINS ASSEMBLE AT THE BREAK, WHEN DOES PROSING OCCUR, WHEN DOES AUTO PHOSPHORYLATION OCCUR, ALL OF THAT BECAUSE A LITTLE BIT DIFFICULT TO UNDERSTAND WITH THIS SEQUENTIAL MODEL FOR NHEJ. SO WE STARTED THINKING ABOUT IT A LOT MORE AS ASSEMBLY OF A MOLECULAR MACHINE AT THE DOUBLE STRAND BREAK AND SO, I THINK THIS CAME ALSO FROM THE WORK FROM DAVID CHAN'S LAB, AND MICHAEL LIEBER'S LAB. THE IDEA THAT KU IS THE FIRST RESPONDER, GETS TO THE BREAK AND CAN RECRUIT MULTIPLE OTHER PROTEINS TO THE BREAK INCLUDING PROCESSING ENZYMES, LIKE THE WELL WORKS SON AND XLF AND ALSO DNA-PK CS AND SO, THIS KIND OF MODEL WAS REALLY STIMULATED BY CONVERSATIONS WITH OUR COLLABORATOR JOHN TAINER SO WE THOUGHT ABOUT IT AS A ASSEMBLY OF THIS MACHINE. STILL, WE'VE GOT THIS PROBLEM, HOW DOES THIS MACHINE ASSEMBLE AND HOW ARE THE DNA ENDS HELD TOGETHER CLOSE ENOUGH TO BE PROCESSED BY ENZYMES LIKE AUTO MUSS AND POLY NUCLEAR TYPE KINASE WITHOUT BEING LIGATED SO WE HAVE TO THINK ABOUT HOW ENSEMBLED AND TETHERED THE ENDS TOGETHER AND PROCESSES THE DNA ENDS AND LIE GATES THEM TOGETHER FINALLY. SO WE BROKE THIS DOWN LOOKING INTO INDIVIDUAL COMPONENTS AND I'LL GO OVER A LOT OF DATA FROM MANY GROUPS IN THE FIELD STARTING WITH THE BEAUTIFUL STRUCTURE OF THE KU DETERMINED DETERMINED BACK IN 2021 WHERE YOU CAN SEE KU80 AND KU70 FROM THIS BASKET STRUCTURE AND THE DNA IN THE CENTRAL PORTION WITH THE POPPY PEPTIDE FORM THIS BEAUTIFUL BASKET STRUCTURE AND THE DNA INTERACTS IN THE ELECTRO STATIC FASHION SO THE KU WITH DOUBLE STRANDED DNA IS NOT SEQUENCE DEPENDENT AND THEN BUILDING ON WORK FROM STEVE JACKSON AND OTHERS SHOWING THAT THE KU80 TERMINAL REGIONS FORMS THIS LONG FLEXIBLE EX TENSE AND IT'S THE EXTREME OF K80 INTERACTS WITH DNA-PK CS SO THIS IDEA THAT KU BINDS TO THE DOUBLE STRAND BREAK IT'S GOT THIS LONG, FLOPPY TAIL THAT BRINGS IN DNA-PK CS TO THE COMPLEX. AND THEN THE BEAUTIFUL STRUCTURE OF DNA-PK CS FROM GROUPS LIKE TOM AND MARTY AND OTHERS SHOW IN THE DNA-PK CS FORMS THIS BEAUTIFUL CIRCULAR STRUCTURE WHERE YOU HAVE THE KINASE DOMAIN AND THE HEAD OR THE CROWN AND THEN THE HEAD REGIONS FORMING THIS BEAUTIFUL ALPHA SOLONIDE AND IF YOU ROTATE IT BY 90¡ YOU CAN SEE AGAIN THE KINASE, THE FACT AND THE FAT AT THE PROTEIN HERE AT THE TOP OF THE HEAD AND THIS TERMINAL HEAT REPEAT REGION FORMS THIS BEAUTIFUL SORT OF CONCAVED SURFACE AND THIS IS THE CRYO STRUCTURE PUBLISHED A FEW YEARS AGO NOW FROM THE CHINESE GROUP AND YOU CAN SEE THAT THE DNA COMES IN THROUGH THE KU AT THE BACKSIDE OF DNA-PK CS AND THIS IS HOW THE DNA-PK CSKU COMPLEX IS FORMED. AND THEN AGAIN, WORK THROUGH MARTY AND WAYNE AND MURRAY AND STEVE JACKSON AND OTHERS DEDUCED THE STRUCTURE OF THE XSCC4 AND IT'S A TWO HEAD DOMAINS AND THE VERY LONG C TERMINAL CALLED COIL REGION AND THIS COILED COIL REGION OF XSCC4 INNER ACTS OF DNA LIE GAZE FORM AND IT'S SIMILAR TO THAT OF XRCC4 IT'S A HEAD DOMAIN AND THESE LONG COILED REGIONS AND IN THE CASE OF XLF THERE'S A TWIST IN THE TAIL IN THE POLY PEP SIDE CHAIN FALLS BACK UP ON IT SO AND WITH JOHN TAINER'S GROUP AND MICHAL HAMEL, IT SHOWS INTERACTS BY THE HEAD DOMAIN IN THIS POCKET. AND SO MANY GROUPS, INCLUDING OURS, HAVE SORT OF TAKEN THESE INDIVIDUAL PROTEINS THAT THE STRUCTURES AND TRIED TO MODEL WHAT THE NON HOMOLOGOUS COMPLEX LOOK LIKE AND THIS MODEL IS TAKEN FROM THE (INAUDIBLE) TO THE DOUBLE STRANDED DNA AND YOU CAN SEE, THERE ARE TWO KU MOLECULES, ONE ON OTHER SIDE AND THEN THIS (INAUDIBLE) INTERACTION WITH HEAD DOMAIN WITH THE DNA LIGATES AND IN THIS MODEL YOU HAVE IT INTERACTING WITH XOCC4 AND THE FLEXIBILITY WITH THE END OF THE DOUBLE STRAND BREAK. I'M GOING TO TAKE A SITE TRACK FOR A MOMENT AND I'LL COME BACK AND TALK ABOUT THE COMPLEX IN MORE DETAIL. SO, SOME OF THE CHALLENGES OF WORKING WITH DNA-PK C ARE ARE ITS SIZE. IT'S VERY BIG. 4,128 AMINO ACIDS, AND YOU CAN SEE FROM THE DIMENSIONS HERE FROM ONE OF TOM'S STRUCTURES, 180 BY 1230 BY 105 SO IT'S A VERY BIG PROTEIN TO WORK WITH AND IT'S DIFFICULT TO MAKE MUTATIONS AND DO SEPARATION OF FUNCTION EXPERIMENTS. AND ANOTHER MAJOR PROBLEM IS, THAT DNA-PK CS OR THE GENE, PRKDC, ALTHOUGH IT'S PRESENT IN HUMANS, MICE, AND ZEBRAFISH, IT'S ABSENT FROM MOST MODEL ORGANISMS AND THE YEASTS AND POMB AND SO THIS IDEA HAS LED TO THE NOTION THAT DNA-PK CS IS A VERVERTEBRAE-SPECIFIC PIK-K DURING THE COVID SHUT DOWN, WE STARTED LOOKING ON THE NCBI DATABASE FOR DNA-PK CS AND WE STARTED TO LINE IN THE SEQUENCES AND AS YOU WOULD EXPECT, WHEN YOU LOOK AT VERTEBRATE, DNA-PK CS HAS A HIGH DEGREE OF AMINO ACID CONSERVATION AND THIS IS BIRDS, FISH, REPTILES, YOU CAN SEE IT'S VERY, VERY HIGHLY CONSERVED AS YOU EXPECT. BUT THEN WE STARTED ADDING IN OTHER SEQUENCES THAT WERE AVAILABLE AND YOU CAN SEE THAT WE WERE PROPOSING THAT THERE'S DNA-PK CS AND ALSO THINGS FROM WORMS, SEA YO URCHANTS AND ONE OF THE MOST SIMPLE FORMS OF MULTI CELLULAR AND INSECTS AND YOU CAN SEE THERE'S A LOT OF AMINO ACID SIMILARITY AND THIS IS WHERE RED MEANS HIGHLY CONSERVED AND BLUE IS LESS CONSERVED AND GRAY IS NOT CONSERVED. SO YOU CAN SEE BY THESE RED LINES, THERE'S A GREAT DEAL OF CONSERVATION IN WHAT IS CALLED THE FOREHEAD DOMAIN OF THE SIMILARITY HERE AT THE. AT THIS POINT YOU MIGHT BE THINKING, WELL, HOW DO WE REALLY KNOW THIS IS DNA-PK CS AND NOT ANOTHER PIKK FAMILY MEMBER? IT TURNS OUT THAT DNA-PK CS HAS UNIQUE FEATURES, NOT FOUND IN THE OTHER PIKKs. ONE IS THIS HEAT REPEAT THAT'S ONLY FOUND IN DNA-PK CS. >> WE ADDED IN DIFFERENT PRKDC SEQUENCES AND WE WERE ABLE TO SHOW SIMILARITIES AT THE AMINO ACID LEVEL RIGHT DOWN TO VERY SIMPLE ANIMALS AND NEMATODE WORMS AND FUNJI, AND POTATO PLIGHT AND MOLD AND EVEN PLANTS AND YOU CAN SEE WHEN WE EXTEND THIS AMINO ACID SEQUENCE COMPARISON TO INCLUDE THINGS LIKE FUNGI, AMIBA, MONTHS AND MOLD, AGAIN, WE SEE THIS CONSERVATION IN THE FOREHEAD REGION, SOME REGIONS OF CONSERVATION THROUGHOUT THE PROTEIN AND AGAIN, AT THE SEA TERMINUS. WE LOOKED AT THESE SEQUENCES, THIS IS JUST TO SHOW THAT WE FOUND DNA-PKcs THROUGHOUT ALL OF THE MAJOR FILER OF AM FIB RANS, REPTILES, FISH, CRABS, BEES, FLIES, CORRAL, FUNGI AND PLANTS AND CURIOUSLY, IT'S NOT IN ALL SPECIES, FOR EXAMPLE, IN THE MENOTODES IT'S IN THE ENOPLEA BUT NOT IN THE CHILD PORNOGRAPHY OWEDOREA AND WHEN WE LOOKED AT THINGS LIKE PLOTS AN -- AND WE LOOKED IN THE FUNGY IT WAS IN THE MICRO HIGH CAR TAR BUT NOT IN THE DIE CARIA YEAST AND IT INCLUDES MANY DIFFERENT PLANTS AND NOT IN THE FLOWERING PLANTS SO IT'S A FLOWERING PLANT AND SO THIS ANALYSIS SHOWS THAT THE INITIAL ASSUMPTION THAT IT'S NOT PRESENT IN SEA AND THE YEAST AND THE FLOWERING PLANTS IS CORRECT BUT DNA-PKcs IS PRESENT THROUGHOUT EVOLUTION IT'S MISSING FROM SOME LINEAGES. OK. SO IT'S NOT A VERTEBRAE-SPECIFIC DNA PIKK. THE OTHER THING WE WANTED TO DO WAS LOOK IN MORE DETAIL AT THE REGIONS OF CONSERVATION AND BY ALIGNING THE AMINO ACID SEQUENCES WE FOUND ONE REGION THAT IS REALLY CLOSE TO THE OR IS RIGHT BETWEEN THE ABCDE PHOSPHORYLATION SITES AND THE WE CALLED IT THE YRPD MOTIF AND THIS IS THE HUMAN SEQUENCE ON TOP. WE HAVE YR THAT IS HIGHLY CONSERVED IN MOST ORGANISMS DOWN TO FUNGI AND PLANTS AND THEN THERE'S A SECOND YR MOTIF THAT'S CONSERVED ALL THE WAY DOWN TO AMIBA PLANTS AND A ONE IS 100% CONSERVED. THIS REGISTER ON WHICH WE HAD WE WHEN WE DIDN'T KNOW WHAT THE FUNCTION WAS IS ABSOLUTELY CONSERVED IN ALL OF THE DNA-PKcs SPECIES THAT WE LOOKED AT. AND OUR COLLEAGUE McHALE HAMMEL AND MODELED WITH THE ABCDE LOOP AND THE YRPD LOOP ARE IN THE DNA-PKcs STRUCTURE SO THE ABCDE PHOSPHORYLATION SITES ARE ON A FLEXIBLE DISORDERED LOOP THAT WE DON'T ACTUALLY SEE IN THE CRYSTAL STRUCTURES BUT MICHAELLE WAS ABLE TO MODEL WHERE HE THOUGHT IT MIGHT BE AND WE HAD SOME DATA SHOW WHERE WE THOUGHT IT WAS LOCATED AND YOU CAN SEE THE ABCDE PHOSPHORYLATION SITES ARE LOCATED RIGHT IN THIS CLEFT OF THIS SOLONIDE AND THE YRPD MOTIF IS RIGHT UP HERE AT THE TOP OF THIS CLEFT IN THE STRUCTURE OF DNA-PKcs. SO THAT IS ALL BEAUTIFUL AND WE'VE GOT ALL THESE BEAUTIFUL STRUCTURES BUT WE STILL DON'T UNDERSTAND HOW THEY ASSEMBLE TO FORM THIS COMPLEX AND THE NHEJ MACHINE. TO START LOOKING AT THIS, WE COLLABORATED WITH DAVID SHA REIMER AT THE UNIVERSITY OF COOLING AND HE HAS DONE A LOT OF BEAUTIFUL WORK USING HYDROGEN-DEUTERIUM EXCHANGE MASS SPECIFIC TOMORROW TEE CROSS LINKING AND HIS STUDENT WAS ABLE TO ASSEMBLE IT ON DOUBLE STRANDED DNA TO FORM AND AND THE TU AND THE DNA AND TO SUMMARIZE WHAT THEY FOUND, THIS IS A MODEL OF THE STRUCTURE WHERE DNA-PKcs IS SHOWN HERE IN THE TEAL AND GREEN AND THE KU IN THE YELLOW AND ORANGE AND THE DNA IS IN PINK. SO WHAT THEY PROPOSED IS THE FORMATION OF THE SYNAPTIC COMPLEX WHERE YOU'VE GOT ONE K ON EACH SIDE OF THE BREAK AND AND THERE WERE TWO REALLY INTERESTING THINGS THAT CAME OUT OF THIS STRUCTURE. ONE WAS, IN THIS STRUCTURE, THE DNA ENDS AND IT'S PROBABLY SEEN BEST IN THIS STRUCTURE, ARE OFFSET BY ABOUT 115 SO THIS IS ONE PIECE OF DNA AND HERE IS THE END. HERE ISED ENTER OF THE DNA AND THE DISTANCE BETWEEN THESE TWO STRANDS OF DNA, ONE POINT IN THIS DIRECTION AND ONE POINTING IN THIS DIRECTION IS ABOUT 115ANGSTROMS AND THIS WAS SEEN FROM A GROUP USING FRET. THEY HAD DETERMINED THAT IN EXTRACTS DNA-PKcs AND KU COULD FORM A SYNAPTIC COMPLEX WHERE THE ENDS WERE HELD IN OPPOSITE DIRECTIONS 115ANGSTROMS APART SO THEY CALLED THIS THE LONG RANGE SYNAPTIC COMPLEX SO THIS STRUCTURE WE GOT FROM MASS SPECIFIC STORM TREE IS THIS LONG RANGE SIN ATTIC COMPLEX AND THE OTHER THING THAT CAME FROM DAVE'S STUDIES WAS THAT REGION OF DNA-PKcs THAT INTERACTS WITH THE END OF OF THE DOUBLE STRANDED DNA. WHAT HE CONCLUDED FROM THIS STUDY WAS THAT THE ABCDE PHOSPHORYLATION SITE LOOP FORMS OF A PLUG THAT ACTUALLY INTERACTS AND BLOCKS THE PASSAGE OF THE DNA THROUGH THE DNA-PKcs MODEL. AND THIS BECAME VERY IMPORTANT LATER ON WHEN WE WERE COLLABORATING ALSO WITH A CRY OWE EM EXPERTS AT NORTHWESTERN UNIVERSITY AND ALAN THOM KIN SON WHO AN EXPERT IN NEW MEXICO. THE IDEA IS TO BRING TOGETHER PEOPLE WORKING IN DIFFERENT ASPECTS OF DNA REPAIR TO SOLVE COMPLEX MULTI-COMPONENT STRUCTURES AND SO, YOU AND ALAN AND I ARE ALL PART OF SBDR AND WE WERE COLLABORATING TO TRY AND SOLVE THE STRUCTURE OF THE SYNAPTIC COMPLEX AND IT WAS DONE BY AN AMAZING PHD STUDENT. THIS IS THE STRUCTURE FROM THE CRYO EM AND I'LL LEAD YOU THROUGH IT IN OLIVE IS DNA-PKcs AND IN PINK IS KU 70 AND KU 80 AND IN BLUE IS THE XRCC4 AND IT INHE CAN AGENTS WITH THE BRTDC AND THESE ARE HELD TOGETHER BY A XLF SO AS I TOLD YOU BEFORE, THEY WILL INTERACT THROUGH THE XOCC IF AND THE DNA COMES THROUGH THE BEAUTIFUL CIRCLE OF CHANNEL I SHOWED AND UP AND INSIDE THE MOLECULE SO WHAT WE'RE CALLING THIS IS A LONG RANGE SYNAPTIC COMPLEX WHERE WE'VE GOT TWO DNA-PKcs KU MOLECULES AND ONE ON EITHER SIDE OF THE BREAK AND THEY'RE HELD TOGETHER IN THIS CONFIRMATION BY AN XOCC4XLF SCAFFOLD. THIS JUST SHOWS THE COMPLEX IN ANOTHER VIEW WHERE I'VE USED THE RAINBOW COLORS. THE BLUE IS THE ENTER TERM INUS AND THE RED IS THE C OF DNA-PKcs THE KINASE DOMAIN SO YOU CAN SEE AGAIN THIS IS THE SAME COMPLEX WHERE KU NOW IS JUST IN THIS LIGHT BROWN COLOR AND HERE IS THE SCAFFOLD WITH XOCC4 LIGATES THROUGH THE HEAT REGION AT THE TOP, IS THE KINASE DOMAIN WITH KINASE THE FACT AND THE FACT C AND IF YOU ROTATE THIS STRUCTURE FOR YOU BY 45¡, CAN YOU SEE AGAIN MORE CLEARLY THE KINASE DOMAIN, THE ONE DNA-PKcs AND YOU CAN SEE THE OTHER DNA-PKcs MOLECULE IS INVERTED AND THEY'RE JUST SNUGGLING TOGETHER WHERE HERE IS THE HEAD DOMAIN, HERE IS THE CRADLE, AND THEY'RE SNUGGLING TOGETHER SO THAT THE KINASE DOMAIN OF ONE PARTNER IS POSITIONED CLOSE TO THE CONCAVED CAVITY OF THE HETERO PETES OF THE OTHER PARTNER. AND THIS JUST SHOWS ANOTHER VIEW. HERE IT'S FLIPPED BY 90¡ IN THIS STRUCTURE AND IF WE TAKEAWAY THE PROTEINS AND WE JUST LOOK AT THE PATH OF THE DNA, THE DNA IS COMING THROUGH KU INTO DNA DNA-PKcs AND IT IS SEPARATED BY 115 SO THIS IS WHAT WAS PREDICTED BY THE GROUP FROM THE FRET AND ALSO BY DAVE SCHRIEMER'S GROUP BY THE MASS SPECIFIC TROLL TREE SO WE'RE CALLING IT A KON A LONG RANGE SIN ATTIC. THIS IS A MOVIE THAT WILL ROTATE SO YOU'VE GOT THE SCAFFOLD AT THE BOTTOM AND IN YELLOW AND BRONZE, THESE ARE THE BRCT REPEATS OF LIE GAZE 4. WE DON'T SEE THE CATALYTIC DOMAIN IT'S BECAUSE FLEXIBILITY AND TETHERED BY THIS FLEXIBLE LINKER. BUT WE SEE THE BRC DOMAINS. THE XRCC4 AND THE XLF AND WHAT WE'RE REALLY INTERESTED IN IS WHAT GOES ON HERE AT THE INTERFACE WHERE THE TWO DNA-PKcs MOLECULES ARE INTERACTING. AND YOU CAN SEE IN PINK, YOU CAN PROBABLY JUST MAKE OUT THE TERMINAL REGION IS RIGHT THERE IN THIS INTERFACE AND WE'RE MISSING MOST OF THE TERM INUS BUT THIS LITTLE BIT RIGHT AT THE C TERM INOUS, THE EXTREME SIX OR SEVEN AMINO ACIDS IS VISIBLE IN THIS STRUCTURE. THE OTHER THING YOU WILL NOTICE IS IT'S A PIECE OF THE SEQUENCE TO THIS ABCDE LOOP AND YOU CAN SEE THERE'S ONE FOREHEAD LOOP HERE AND HERE IS THE OTHER LOOP FROM THE PARTNER DNA-PKcs AND HERE IS THE INTERFACE LOOP FROM ONE PARTNER AND THE INTERFACE LOOP FROM THE OTHER PARTNER SO WE THINK THESE TWO DNA-PKcs MOLECULES ARE ASSOCIATED ACROSS THIS SYNAPSE. AS I MENTIONED, THESE REGIONS ARE HIGHLY CONSERVED AND THIS SHOWS THE CONSERVATION IN THE FOREHEAD LOOP AND THIS SHOWS THE CONSERVATION IN THE DIMERIZATION LOOP AND THOSE GOING DOWN TO SPONGE AND PLANNE HIGHLY CONSERVED IN THAT REGION. THE MOST BEAUTIFUL PART OF ALL IS WHEN WE LOOK IN A LITTLE BIT MORE DETAIL, HERE IS OUR DIMERIZATION ROOM AND HERE IS THE YRPD MOTIF AND THIS YRPD MOTIF IN THIS STRUCTURE FORMS A LITTLE BETA SHEET AND IT EXISTS IN COMPLEX WITH AN ANTI PARALLEL BETA SHEET FROM WHAT WE'VE CALLED THE YRPD INTERACTING MOTIF SO THESE FORM THIS LOOP WITH THE YRPD AND THE YRPD EDGER INTERACTING MOTIF AND WE HAVE THE UNSTRUCTURED LOOP WITH THE PHOSPHORYLATION SITES AND WE HAVE WHAT WE'RE CALLING THE DNA END BINDING HELIX SO THIS IS A MALL HELIX HERE WE CALL DNA END BINDING APPENDED AND THERE'S A LITTLE LINKER AND THERE'S 37 AMINO ACID HELIX THAT IS INTERACTING DIRECTLY WITH THE ENDS OF THE DNA. THERE'S 300 AMINO ACIDS IN THE ABCDE PHOSPHORYLATION SITES UP UNTIL THE BEGINNING OF THE FAT DOMAIN JUST CONTAINS THIS REALLY BEAUTIFUL REGULATORY SORT OF UNIT AND THE DIME A LOOP, THE YRPD INTERACTING LOOP, THE AUTO PHOSPHORYLATION SITES, AND THEN THIS HELIX THE DEBA LINKER AND FOLLOWED BY THE HIGHLY CONSERVED YRPD MOTIF AND THE FAT DOMAIN. AND THIS IS JUST ANOTHER VIEW OF THAT AND HERE IS THE DNA END BINDING HELIX SHOWN HERE. HERE IS THE DOUBLE STRANDED DNA. YOU CAN SEE THE YRPD AND THE YRPD INTERACTING MOTIF IN YELLOW AND RED AND IN MARINE, IT'S THE DIMERIZATION LOOP AND IN GROWN IS THE FOREHEAD LOOP SO THIS IS LIKE ONE SIDE. AND THIS IS THE OTHER ONE AND THEY SWITCHED AS I TOLD YOU BEFORE AND THEY SORT OF (INAUDIBLE). AND HERE IS JUST ANOTHER VIEW. AGAIN, JUST SHOWING THE YRPD AND THESE REGIONS THAT WE THINK ARE IMPORTANT FOR THIS INTERFACE. AND I'VE TOLD YOU THE YRPD IS HIGHLY CONSERVED AND THE DIMERIZATION LOOP IS HIGHLY CONSERVED AND THERE'S SOME CONSERVATION IN THE YRPD INTER ACTING MOTIF AND THE DEBA MOTIF, THAT LITTLE HELIX, HAS HIGHLY CONSERVED BASIC AMINO ACIDS. WHEN WE LOOKED IN THE LARGE HELIX, THE 37 AMINO ACIDS HELIX, THERE WASN'T SO MUCH ABSOLUTE AMINO ACID CONSERVATION BUT IF WE LOOK AT THE LARG CHARGE, THE CHARGE IS CONSERVED SO WE THINK THIS DNA END BINDING HELIX IS FORMING THIS POSITIVITY CHARGED REGION THAT IS INTERACTING WITH THE DNA. AND SO, IF WE PUT ALF THIS TOGETHER, WE CAN COME UP WITH A MODEL FOR HOW WE THINK THIS DIMERIZATION INTERPHASE REGULATES AT OWE PHOSPHORYLATION INDUCED ASSOCIATION. SO, FOR THE CATALYTIC SITES TO ACTUALLY BE IN CLOSE ENOUGH CONTACT WITH THE ABCDE LOOP, WE THINK THAT THE FIRST NEEDS TO BE A CONFIRMATIONAL CHANGE THAT WOULD DISRUPT THIS YRPD, YRPDI INTERACTION AND THIS WOULD ALLOW AUTO PHOSPHORYLATION OF THE ABCDE LOOP IN TRANS ACROSS THE SIGSIGN APPS AND THE INTERACTION BETWEEN THE DNA END BINDING MOTIF AND THE DNA LEADING TO THE RELEASE OF DNA-PKcs FROM THE DNA END AND THE SYNAPTIC COMPLEX. SO THAT WOULD BE THE MODEL TO EXPLAIN WHAT WE THINK IS GOING ON WITH THE AUTO PHOSPHORYLATION PHOSPHORYLATION. OF SITES ARE IN PROXIMITY TO THE PQR CLUSTER AND THE ABCDE CLUSTER OF THE OPPOSITE DIME A. I WANT TO TALK ABOUT THE SECOND COMPLEX THAT GENERATE AND THIS WAS GENERATED IN THE ABSENCE OF DNA-PKcs. SO, AGAIN, WE HAVE THE XLF, HOME A DIME A. WE HAVE THE XRCC4 THROUGH THE HEAD DOMAIN AND WE HAVE THE DNA WITH THE LONG C TERMINAL STOCK ON XRCC4 AND IT'S INTERACTING WITH THE KU BUT YOU CAN SEE THAT NOW THIS COMPLEX IS UNDERGONE A CONFIRMATIONAL CHANGE WHERE IT'S LIKE THE KUs HAVE SWIVELED AND THE XRCC4s HAVE SWIVELED AND NOW THE DNA ENDS THAT WERE IN CLOSED IN THE DNA-PKcs ARE NOW FREE AND NOW THEY ARE PARALLEL TO EACH OTHER -- HORIZONTAL I SHOULD SAY, WITH THE ENDS' POSITIONED FOR LIGATES. AND SO WHAT WE ARE ALSO ABLE TO SEE IN THIS SHORT RANGE COMPLEX, SO WE ASSEMBLED ON THIS DOUBLE STRAND BREAK. AND THIS IS A MOVIE AND IF I CAN DO IT. WE HAVE XRCC4 AND XLF AND THE DNA IS PARA LEGAL AND WE SEE A SINGLE CAT A LID I CAN AT THE BREAK AND THIS IS IMPORTANT -- SORRY, BECAUSE I GET TO THAT, SO THIS WOULD BE A MODEL THEN FOR HOW WE COULD GO FROM TRANSITIONS FROM THIS LONG RANGE COMPLEX, TO A SHORT RANGE COMPLEX AND THIS IS JUST A LITTLE MOVIE AND IT HOPEFULLY I CAN SHOW HER HERE IT HERE. HERE WE GO. SO HERE IS THE DNA DRAWN UP INSIDE THE DNA-PKcs. YOU GET YOU AUTOPHOSPHORYLATION AND DIS ASSOCIATION AND YOU GET A CONFIRMATIONAL CHANGE THAT BRINGS THE ENDS OF DNA CLOSE ENOUGH TO BE LIGATE LIE GATE GAYS. YOU SHOULD SEE ILIGATED.SO THERE'S ONE MOR E LITTLE PIECE TO THIS PUDDLE AND THAT IS DNA LIE GAZE 4 ONLY CATALYZE REPAIR OF ONE NICK OR SINGLE STRAND BREAK. THE SECOND LIGATES COMES INTO SEAL THE OTHER END. IT REPAIRS THE NICK AND THEN THE OTHER MOLECULE, THE OTHER LIGASE 4 WILL COME IN AT SOME POINT, TO SEAL THE SECOND NICK. THERE WE GO. SO THAT IS THE MODEL FOR HOW WE THINK THAT COMPLEX WORKS. SO THIS JUST SUMMARIZES AGAIN WHERE WE'VE GOT THE KU BIND INTO THE ENDS OF THE DNA. KU RECRUIT TRANCE LOCATED INWARDS ABOUT 15 BASES TO ALLOW FOR RECRUITMENT OF DNA-PKcs AND DNA-PKcs NOW OCCUPIES THE EXTREME END OF THE DNA WHICH IS SOMETHING THAT BILL DINEN FOUND IN 1999 AND THIS DNA GOES INSIDE THE DNA-PKcs, IT'S BLOCKED BY THE ABCDE LOOP AND THE DNA END BIND IN HELIX. YOU GET AUTOPHOSPHORYLATION OF THAT ABCDE LOOP ACROSS THE SIGN APPS AND OF COURSE I FORGOT TO MENTION, THAT WE'VE ALSO GOT THE XRCC4 LIGASE COMING IN AND DIS ASSOCIATES AND THEN YOU GET THIS SEQUENTIAL LIGATION. I THINK THAT'S ALL THAT I HAD TO SAY. I WON'T GO THROUGH THAT AGAIN. I JUST SAID ALL OF THAT SO THE FIRST SUMMARY IS THE STRUCTURE OF THE LONG-RANGE SIN ATTIC COMPLEX AND THE SHORT RANGE SYNAPTIC COMPLEX AND A MODELING FOR HOW THE TRANSITION MIGHT OCCUR FROM ONE TO THE OTHER THROUGH THIS AUTOPHOSPHORYLATION INDUCED ASSOCIATION AND THE SECOND POINT THAT I REALLY WANT TO LEAVE YOU WITH, WE REALLY THINK DNA-PKcs IS NOT VERTEBRAE SPECIFIC AND IT'S PRESENT IN MANY, MANY DIFFERENT ORGANISMS I THINK IT'S HIGHLY CONFIRMED THROUGH EVOLUTION AND IN PARTICULAR THESE MOTIFS WE'VE IDENTIFIED ARE CONSERVED SUGGESTING THE FUNCTION OF DNA-PKcs AS A END BINDING COP PLEX IS CONSERVED THROUGH EVOLUTION SO WE SEE DNA-PKcs AS A MOLECULAR RULER WHERE IT DRAWS THE DNA INTO THE DNA-PKcs SO THAT THE END IS TEETHER THE AT THE SIN ATTIC COP PLEX. THEY CAN GET PROCESSED AND THE DNA-PKcs IS REMOVED AND THEN LIGATION CAN OCCUR. AND THAT IS ALL I WAS GOING TO SAY. I'D LIKE TO THANK THE PEOPLE IN MY LAB AND MY COLLABORATORS AND CALL A FIELD OWES HIM A GREAT DEBT OF GRATITUDE FOR THE WORK HE DID AND IT'S SAD HE PASSED AWAY SO SOON. >> MY LAB MEMBERS AND JOHN McHALE AND SUSAN CAFFY A WONDERFUL FRIEND AND DAVE SCHRIEMER AND MY HUSBAND JAMES IS INVOLVED BECAUSE OF COVID, HE DIDN'T HAVE A LOT TO DO AND HE LIKES EVOLUTIONARY BIOLOGY, IT'S A HOBBY SO HE WAS VERY MUCH INVOLVED IN THE IDENTIFICATION OF THE CONSERVED REGIONS OF DNA-PKcs. AND I DIDN'T HAVE TIME TO TELL YOU TODAY, BUT WE'VE ALSO HAD A REALLY NICE COLLABORATION WITH TERRANCE STRICT IN PARIS, DOING SINGLE MOLECULE EXPERIMENTS THAT REALLY LED TO SOME OF THE IDEAS THAT CAME THROUGH IN THIS SEMINAR AND OF COURSE FUNDED THROUGH THE SBDR INITIATIVE AND NIH GRANT TO UN AND ALSO THE CANADIAN NATURAL SCIENCES AND ENGINEERING AND RESEARCH COUNCIL. IF YOU'VE NEVER BEEN TO CALGARY, THIS IS DOWNTOWN. THIS IS NICE. IT'S NOT TODAY BUT HOP HOPEFULLY SPRING WILL BE HERE SOON. IF ANYONE IS INTERESTING IN WORKING ON DNA-PKcs, PLEASE, GIVE ME AN E-MAIL AND I'D LOVE TO CHAT WITH YOU. THANK YOU, VERY MUCH. >> THANK YOU, VERY MUCH FOR A WONDERFUL TALK, SUSAN AND WE REALLY ENJOYED IREALLY ENJOYED IT. I'LL START READING THE FIRST QUESTION HERE FROM CHRISTINA. IT SAYS HOW MANY SITES IN THE ABCDE LOOP ARE NEEDED TO BE PHOSPHORYLATIONED IN ORDER TO SUPPORT DOUBLE STRAND BREAK REPAIR AND ALSO WHAT IS THE ROLE OF THE DNA-PKcs IN ANTIVIRAL IMMUNE AND AUTOIMMUNE DISEASE? >> KATHY MADE INDIVIDUAL MUTATIONS WHEN WE FIRST DISCOVERED AND WE MADE GROUPS OF MUTATIONS. WHEN WE MADE THE INDIVIDUAL MUTATIONS IN THE PHOSPHORYLATION SITE, THERE WAS ONLY A SMALL DEFECT IN DNA DOUBLE STRAND BREAK REPAIR WITH SENSITIVITY TO RADIATION SO IT SEEMS THAT YOU NEED TO GET MORE THAN ONE OF THOSE SITES PHOSPHORYLATEDDED IN ORDER TO SEE THE EXTREME RADIO SENSITIVITY WE SEE WITH THE ABCD ABCDE MUTANT. MOST ARE PHOSPHORYLATEDDED IN RECEIVE OWE. IF YOU LOOK AT THE SITE, THEY'VE BEEN SHOWN TO BE PHOSPHORYLATEDDED IN VIVO. THERE'S AT LEAST 40 INVITO SITES WE HAVE IDENTIFIED AND THERE'S AT LEAST 100 POST TRANSLATIONAL BEING IDENTIFIED ON DNA-PKcs SO PROBABLY GETS VERY COMPLICATED AND I DON'T HAVE THE ANSWERS TO THAT. THE OTHER QUESTION WAS ABOUT THE AUTOIMMUNE -- KU WAS IDENTIFIED AS AN AUTOIMMUNE ANTIGEN IN PATIENTS WITH AUTOIMMUNE DISEASE AND THAT'S WHAT LED TO ITS PURIFICATION AND ITS CHARACTERIZATION. SO I GUESS AT THAT TIME THE LINK BETWEEN DOUBLE STRAND BREAK REPAIR AND IMMUNE RESPONSE WERE NOT CLEAR, APART FROM OBVIOUSLY VDJ ADAPTIVE IMMUNE RESPONSE BUT MORE RECENTLY THERE'S MORE AND MORE EXAMPLES OF DNA-PKcs AND KU BEING INVOLVED IN INNATE IMMUNITY AND DETECTION OF DOUBLE STRANDED DNA FRAGMENTS, I GUESS, IF YOU WILL IN THE SITE OWE PLASM AND AND IT'S THROUGH SMALL MARGMENTS OF DNA. >> CAN YOU STOP SHARING YOUR SCREEN PLEASE, SO WE CAN SEE YOU, SUSAN. SIGHT OWE PLASM. WHAT IS QUALITY CONTROL FOR DEALING WITH THOSE AND PERHAPS SOME -- >> FANTASTIC QUESTION. ABSOLUTELY. SO, RADIATION INDUCES A WHOLE BUNCH OF DNA DAMAGE. DAMAGE TO THE ENDS OF THE DNA, DAMAGE SURROUNDING THE DOUBLE STRAND BREAK AND THAT HAS TO BE REPAIRED OR THE NON LIE GATE ABLE GROUPS HAVE TO BE REMOVED PRIOR TO LIGATION SO I GUESS WHAT WE SEE IS THAT THE ENDS ARE PROTECTED SO THEY'RE NOT INADVERT ANT BUT THERE HAS TO BE A HAND OVER TO BETWEEN DNA-PKcs AND THE PROCESS IN END SYMMES SO AUTO MUSS INTERACTS WITH DNA-PKcs AND LIGASE 4 SO IT'S ONE CANDIDATE THAT MAYBE COULD COME IN AND HELP REMOVE SOME OF THOSE NON LIGATABLE END GROUPS AND PNK INTERACT WITH XRCC4 AT SOME POINT, I THINK, YES, THESE SYNAPTIC COMPLEXES THAT WE'VE DEFINED HAVE TO COORDINATE WITH THE PROCESS IN ENZYMES SO THEY'RE REMOVED AND ONLY THEN CAN THE LIGASE CARRY OUT THE FINAL LIGATION STEP. HOW THAT HAPPENS, I DON'T KNOW. I THINK IT'S GOING TO INVOLVE PHOSPHORYLATION AND I THINK IT'S GOING TO INVOLVE A.T.M. THE BACK STORY TO MY TALK IS, I SPENT MOST OF MY CAREER IDENTIFYING INVITO DNA-PKcs PHOSPHORYLATION SITES THAT TURNED OUT TO BE PHOSPHORYLATEDDED BY A.T.M. THEMSELVES. [LAUGHTER] SO AND IT PHOSPHORYLATION AND I'M SURE PHOSPHORYLATION BY OTHER ENZYMES IS AS WELL AS DNA-PKcs IS GOING TO BE IMPORTANT IN REGULATING THAT TRANSITION. DON'T KNOW THE ANSWER YET. WE HAVE A VERY FROM BEVERLY MUCK, WHAT CAUSES THE RELEASE OF DNA-PKcs FROM THE COMPLEX? >> SO, WE'VE DONE SOME SMALL CAPACITORRING EXPERIMENTS WITH JOHN THAINER'S LAB WHERE WE LOOKED AT THE SHAPE BEFORE AND AFTER AND SO, I THINK THAT THAT'S PART OF IT AND THE AUTOPHOSPHORYLATION IS GOING TO DISRUPT THE INTERACTION WITH THE ENDS OF THE DNA BECAUSE THE NEGATIVE CHARGE OF THE PHOSPHORYLATION WILL COMPETE WITH THE DNA END BINE AND THEN, I GUESS THAT CONFIRMATION WILL CHANGE JUST CAUSE TS TO DISASSOCIATE. THE TERMINAL REGION IS IN THAT INTERPHASE 2 SO THERE COULD BE CHANGES THAT AFFECT ITS INTERACTION WITH THAT LITTLE MOTIF THAT RECEIVE JACKSON HAS SHOWN IS REQUIRED FOR INTERACTION OF KU WITH DNA-PKcs SO I IMAGINE IT WOULD BE A LOT OF CONFIRMATIONAL CHANGES AT THAT INTERFACE THAT ARE GOING TO BE TRIGGERED BY THE AUTOPHOSPHORYLATION. >> THANK YOU. THERE'S A QUESTION FROM STEVEN LLOYD. WONDERFUL PRESENTATION. IF THE ENDS OF THE DNAs CANNOT BE LIGATED BY SEQUENTIAL ACTIVITIES OF LIGASE 4, IS THERE FURTHER END PROCESSING OR IS THIS CHECKED PRIOR TO THE FINAL ASSEMBLY COMPLEX? >> FANTASTIC QUESTIONS. I DON'T KNOW THE ANSWER IT THAT EITHER. WE KNOW THAT IN VDJ RECOMBINATION, USING MODEL DOUBLE STRAND BREAKS, THERE'S LOTS OF ABOUT SIX TO 10 BASIS CAN CURE ON EITHER SIDE OF THAT DOUBLE STRAND BREAK AND SO I THINK THIS IDEA OF THE HAND OVER BETWEEN THE PROTECTION AND THE TEETHERRING BY DNA-PKcs HANDING OVER TO THOSE PROCESSING ENZYMES THAT'S GOING TO BE THE CRUCIAL STEP. YOU GET THIS NUCLEOTIDE LOSS SO I THINK THAT'S PROBABLY WHY NEHJ IS SEEN AS BEING ERROR PRONE BECAUSE THE HAND OVER IS NOT PERFECT. YOU DON'T GO FROM THIS PROTECTED ENDS AND THEN THE ENDS OF PROTECTED TO BE IN LIGATED WITHOUT ANY PROBLEMS, THERE'S GOT TO BE SOME GAP IN SPACE IN THERE FOR THE END GROUPS TO BE REMOVED AND PROCESSING TO BE OCCURRED AND NUKELY ACES TO GET IN AND DUE PROCESS IN. THAT'S THE NEXT STEP IS UNDERSTANDING EXACTLY HOW THAT TRANSITION GOES AND THE HAND OVER TO THE PROCESS AND END SYMMES. BUT SORRY, I'M HAND WAVING BECAUSE I DON'T HAVE AN EXPLANATION. >> THERE'S AN E-MAIL HERE FROM EVE AND HE SAYS HE COULDN'T CONNECT TO THE SYSTEM BUT HE WANTS TO, HE SAYS, BEAUTIFUL WORK AND I ENJOYED YOUR PRESENTATION AND YOUR COMMITMENT TO THE SEDATION OF THE NEPK IS OUTSTANDING AND IT'S CONNECTION TO TOP ONE AND TOP TWO BREAK ROW PAIR REMAINS TO BE CLEARLY DISSECTED RADISSECTED AS IT'S EXTREMELY DIFFERENT. REPAIRS TO TOP 2 VIA DNA DAMAGE FOR TOP 1. DO YOU HAVE ANY COMMENTS ON TOP 1 AND TOP 2 HERE? >> YEAH, THAT'S A FANTASTIC QUESTION. YOU GET A CO VEIL ANT INTERACT BETWEEN THE END OF THE DNA AND THE SUMMARY AND IT HAS TO BE REMOVED SO AGAIN THERE'S GOING TO BE SOME FANTASTIC SORT OF INTERACTIONS AND COORDINATIONS GOING ON THERE BETWEEN THE TOP SUMMER AND THE END JOINING COMPLEX. I DO NOT KNOW HOW IT OCCURS BUT IT'S A FANTASTIC QUESTION AND SOMETHING THAT NEEDS TO BE EXAMINED. THE TIME AS COME TO A CLOSE AND I REALLY WANT TO THANK YOU SO MUCH FOR THIS WONDERFUL PRESENTATION AND I'M FASCINATED BY THE WAY YOU STRUCTURAL BIOLOGISTS WORKING AT THE FINEST LEVEL OF THE MOL HE CAN EYE LAR MOLECULAR ARE USING HAND-WAVING GESTURES TO SHOW HOW THESE MOLECULES INTERACT AND IT'S REALLY WONDERFUL WAY OF COMMUNICATING THESE VERY COMPLEX ISSUES. THANK YOU FOR THAT. [LAUGHTER] >> THANK YOU SO MUCH. >> WE SHOULD DO MORE OF THIS. >> THANK YOU. WE REALLY ENJOYED. >> THANK YOU, EVERYBODY FOR LISTENING. >> THANK YOU. AND THANK YOU FOR THE GREAT QUESTIONS. >> BYE.