HELLO THERE, EVERYONE.
WE HAVE A VERY FULL AGENDA TODAY
SO I WILL TRY TO BE BRIEF MUCH
WE HAVE AN EXCITING PROGRAM
LINED UP FOR YOU IN THE FORM OF
3 STELLAR YOUNG INVESTIGATORS
WHO HAVE BEEN NOMINATE BIDE OUR
SCIENTIFIC ADVISORY BOARD.
BUT BEFORE I HAND THINGS OVER TO
DR. MCINTOSH WHO WILL INTRODUCE
THE FIRST SPEAKER, I WANT TO
REMIND TO YOU PUT YOUR QUESTIONS
IN THE CHAT BOX.
OVER TO YOU EMPLOY
>> THANK YOU, KAREN.
GOOD AFTERNOON, EVERYONE.
I'M DELIGHTED TO INTRODUCE OUR
FIRST SPEAKER OF THE SESSION,
TODAYING SPECIES AND D, INA
REPLICATION IN THOSE SPECIES,
FOLLOWING WHICH SHE DID A POST
DOCTORAL FELLOWSHIP WITH
DR. SAMUEL WILSON AT THE NIEHS,
INVESTIGATE BASIC MOLECULAR
MECHANISMS RELATED TO BASIC
ECCISION REPAIR AND THEN IF 2019
SHE WAS RECRUITED BY THE
UNIVERSITY OF FLORIDA TO START
AN INDEPENDENT CAREER AS A
TENURED TRACK ASSISTANT
PROFESSOR IN THE DEPARTMENT OF
BIOCHEMIST RADIOY AND MOLECULAR
BIOLOGY.
IN HER INDEPENDENT POSITION, SHE
CONTINUES TO STUDY THE PATHWAY
PARTICULARLY LOOKING AT THE
INTERPLAY BETWEEN THE DNA
POLYMERASE BETA OR LIGASE 3 OR
1A, NOW HER WORK HAS BEEN FUNDED
BY A K99, R00 AS WELL AS MOST
RECENTLY AN R35 EARLY STAGE
INVESTIGATOR GRANT FROM THE
NIGMS AND I'M LOOKING FORWARD TO
HEARING HER TELL US ABOUT HER
RECENT WORK AND THE TITLE OF HER
TALK IS NEXT STOPS ON THE ROAD
TO DNA REPAIR EMPLOY NTHANK YOU
SO MUCH FOR THE NICE
INTRODUCTION AND HELLO EVERYONE
EMPLOY I REALLY APPRECIATE THE
OPPORTUNITY AND IT'S A GREAT
PLEASURE TO PRESENT MY WORK TO
THE GROUP.
SO LET'S TART WITH THE
DMA LIGASES, YOU KNOW VERY WELL,
YOU KNOW DNA CAN BE GENERATED
DURING NORMAL DECKER NA
TRANSACTION OR DNA LESION BY
EXCISION REPAIR AND THERE ARE
POTENTIALLY--BY JOINING THE
PRIME ENDS.
THEREFORE D NA LIGASES ARE A
FUNDAMENTAL ENZYME TO MAINTAIN
THE STRUCTURAL
ENTEGGIC--STRATEGICRITY OF THE
HUMAN GENOME.
PARTICULARLY THEY FINALAISE ALL
DNA REPAIR PATHWAYS AND CLAIM
IMPORTANT ROLE FOR THE
[INDISCERNIBLE] LIGANDS WITH THE
NUCLEACE REPLICATION.
SO BASIC EXCISION REPAIR IS THE
DNA REPAIR MECHANISM IN THE
SIMILAR DEFENSE NEAR REMOVING
SMALL AND OFFENSE
NON[INDISCERNIBLE] BASAL LESIONS
SUCH AS OXIDATION AND
ONCUALATION PRODUCTS AS WELL AS
SINGLE STRAND [INDISCERNIBLE].
AND STEPS PROCEEDING IN AN
ELDERLY MANNER AND HE RECEIVED A
SHORT PATCH OR THE SINGLE BASIC
EXCISION PAIR PATH, WHICH ARE
RESPONSIBLE FOR THE REPAIR OF
SINGLE DNA UNTIL [INDISCERNIBLE]
AT THE DNA AT THE BEGINNING HERE
AND THE BASIC EXCISION OF THE
PATHWAY IS INITIATED BY SPECIFIC
DNA GIVING--YOU COLSIS, MAYBE
REMOVAL OF DECKER NA BASE
SOLUTION, WHICH RESULTS IN THE
FORMATION OF [INDISCERNIBLE] AND
A PIECE SIDE AND THE CANNED WHAT
IN A DOUBLE STRANDED DNA AND THE
NEXT ENZYME, WITH THE APP AND 1
AP1 [INDISCERNIBLE] BOUND AT
LEAST TO THE RIBOPHOSPHATE BETA
5 PRIME [INDISCERNIBLE].
AND THEN DNA POLYMERASE IS A
MAJOR CANNED WHAT POLYMERASE
COME WITH A 2 CRITICAL FUNCTION
FIRST THE POL REMOVES IT BY THE
[INDISCERNIBLE] DNA LIGASE
ACTIVITY AND THEN INCORPORATES
THE [INDISCERNIBLE] TO FILL THE
GAP DURING THE DNA SYNTHESIS TO
REPAIR THE PATHWAY AND THIS
CREATES [INDISCERNIBLE] IS A
SUBSTRATE FOR THE NEXT STEP OF
THE REPAIR PATHWAY WITH A 2
PRIME AND 5 PRIME SEALED BY THE
DID, NA LIGASE ALPHA AS A LAST
TEP TO COMPLETE THE PATHWAY.
AND THIS REPAIR PATHWAY
COORDINATION IS FACILITATED BY
MULTITELEPROTEIN, PROTEIN
INTERACTION ON PROTEIN
COMMUNICATION.
IT HAS ALSO BEEN SHOWN IN
[INDISCERNIBLE] IS POST DOC
THERE IS A SUBSTRATE PRODUCT
CHANNELING BETWEEN THE BR
PROTEINS THAT INVOLVES THE
HANDOFF OF REPAIRING
[INDISCERNIBLE] FROM 1 STEP TO
THE NEXT IN THAT SEQUENTIAL
MANNER IN OTHER WORDS PRODUCT OF
EACH STEP SERVE AS A SUBSTRATE
FOR THE NEXT ENZYME IN THIS
REPAIR PATHWAY.
AND IT'S BEEN SHOWN ON THIS
SUBSTRATE CHANNELING MECHANISM
PREVENTS THE RELEASE AND
ACCUMULATION OF THE SINGLE
STRAND BREAK FOR THE CELL AND
PERSIST OF THIS COULD TRIGGER
SOME CELL CYCLE AND HOPEFUL
NUCLEAR RECOMBINATION ACTIVITIES
AND IN ADDITION TO THAT BR AND
CO-BR PROTEINS I'M SHOWING HERE
AND IT'S BEEN KNOWN UNDER SOME
NONENZYMATIC SCAFFOLDING FACTORS
SUCH AS [INDISCERNIBLE]
ESSENTIAL FOR A PATIENT BR AND
EXCIDE CC1 PLAY A ROLE IN THE
PROMOTER REPAIR AND THE
TREATMENT AND OF THE DNA DAMAGE
AND PREVENTING THE FROM BEING
RELEASED AND THE INDIVIDUAL
STEPS HERE JUST STILL ON THE
RULING TO HOLD THEM OUT TO THE
BR COMPLEX, FOR THE REPAIR
PATHWAY COORDINATION.
AND THE DOWN STREAM STEEPS
INVOLVING PATHWAY INVOLVING GUT
FEELING AND POL-BETA AND
DNALIGASE 1 AND 3 ALPHA AND
EXCHANGEABLE BETWEEN THE
[INDISCERNIBLE].
AND REGARDING THE POLBETTA, IT'S
THE SMALL [INDISCERNIBLE] DNA
POLYMERASE, INCLUDING
[INDISCERNIBLE] AND THE
POLYMERASE DOMAIN FOR 3
SUBDOMAINS, DNA BINDING, AND
DNATPs, AND YOU CAN SEE IT IN
THE STRUCTURAL MODEL OF THE
PROTEIN.
IT'S BEEN SHOWN BEAUTIFULLY BY
[INDISCERNIBLE] OF HUMAN PERSON
MUTATIONS CARRY THE PON, L-GENE
AND IT CAN BE ASSOCIATED WITH
VARIANTS IN MOUSE CELLS
RESULLING IN PERMANENT CELL
TRANSFORMATION [INDISCERNIBLE]
TOXIC [INDISCERNIBLE].
REGARDING THE DNA LIAISON CASES,
THEY SHARE HIGHLY CONCERTED
C-TERMINAL, FOR THE BINDING
DOMAIN AND THE OTHER
[INDISCERNIBLE] DOMAIN, END
TERMINAL TO THE CAN THEA LYTIC,
THERE'S A BINDING DOMAIN THAT
BINDS MORE ROBUSTLY AND THE
CATALYTIC CORE IS BINDED BY AN
ACTIVE SEAT RESULTING IN THIS
CORE AND CATALYTIC REAMINGION
EXTENDS A CONFIRMATION IN THE
ABSENCE OF DTHRKA, AND LACK
CONFIRMATION CHANGE OF CURES
WITHIN THE CORE THAT BINDS TO
THE DNA BINDING DOMAIN TO FORM
LIGASE PROTEIN CLAMP LIKE
ARCHITECTURE WHICH CIRCLES THE
[INDISCERNIBLE] WHICH YOU CAN
SEE THIS STRUCTURE FOR BOTH DNA
LIGASES.
AND BOTH LIGASES HAVE A BLANKING
UNRELATED END TERMINAL AND
C-TERMINAL REGIONS AND
DURING--THEY PLAY AN IMPORTANT
PART IN THE SPECIFIC
PROTEIN-PREEN TO INTERACTIONS
WITH THE OTHER PROTEIN AND DNA
REPAIR REPLICATION PROTEINS AND
SUBSEQUENT LIGASES AND FUNCTION
AND TO PARTICIPATE IN THE
CELLULAR FUNK.
TREAT END TERMINAL DOMAIN LIKE
IS 1 THAT ACTS WITH THE PCA AND
OTHER PROTEINS, SAME THING FOR
INTERACTION BETWEEN LIGASE 3
ALPHA AND P A RP1 EMPLOY AND
IT'S BEEN KNOWN THAT THE SARS
KRRK CWOBDERFUL--WONDERFUL MAKES
A [INDISCERNIBLE] OFF LIGASE 1
THAT OPERATING TO THE
[INDISCERNIBLE] HISTORY
SUBCELLULARLY.
AND LIGATION REACTION IS ALSO
HIGHLY CONSERVED.
THIS USES A FRACTOR FOR THE
REACTION INVOLVING 3 SUBSEQUENT
CHEMICAL STEPS AND DURING STEP 1
AND ON THE ATP LIGASE RESULTS IN
THE FORMATION OF THE FIRST
INTERMEDIATE LIGASE AMP.
THIS IS LISTENED TO THE ACTIVE
RESDUE RESIDING IN THE
DOMINATION OF THE LIGASE AND YOU
SEE THE [INDISCERNIBLE] 568 FOR
LIGASE 1.
AND DURING THAT STEP TO THE
AMPOT LIGASE IS TRANSFERRED TO
THE 5 PRIME, 5 PRIME END TO THE
[INDISCERNIBLE] AND FORMING
ANOTHER LIAISON GAGE REACTION
TERM DNA, [INDISCERNIBLE] AND
DURING THE LAST STEP 3 OFF THE
LIGATION REACTION,
[INDISCERNIBLE] ATTACKS THE
[INDISCERNIBLE] DOWN STREAM TO
JOIN THIS 3 PRIME WHICH IS
COUPLED TO THE AMP RELEASE.
AND THIS LIGATION REACTION
REQUIRES THIS WAD WHAT.-
-[INDISCERNIBLE].
AND WE KNOW THAT THIS MAY FEEL
IN THE DAMAGE OR MODIFIED 3
PRIME EPPED RESULT NOTHING THE
FORMATION OF LIGATION FAILURE
PRODUCTS.
AND THOSE LIGATION PRODUCT
[INDISCERNIBLE] AND IT'S
ACTUALLY LESIONS, AND THEREFORE
IT'S FORMATION OF THE REPAIR OF
[INDISCERNIBLE] ARE EXPECTED TO
BE CRITICAL IN LIABILITY AND
GENOME STABILITY.
THERE ARE SOME ENZYMES AND
PROCESSING TO FIGHT TO CARE OF
THAT LIGATION PRODUCTS SUCH AS
APT AND TOXIN.
IT HAS BEEN SHOWN BEAUTIFULLY BY
[INDISCERNIBLE] THAT THE LIGASE
1 IS COVERED BY THE
[INDISCERNIBLE] BY USING BINDING
SITE AND SUBSTRATE TO RELEASE A
[INDISCERNIBLE] DNA MIX.
AS I SAID BEFORE, IN MY LAB
STUDIES, DOWN STREAM STEPS ARE
THOUGHT TO BE OUR
[INDISCERNIBLE] BY GAP FILLING
SUBSEQUENT [INDISCERNIBLE] AND
POL BETA AND DNA LIGASES
COORDINATE TO EFICIALLY PASS
THROUGH THE INTERMEDIATE JUST
LIKE INCOMING RUNEDDER VERSE US
THE OTHER [INDISCERNIBLE].
AND WE KNOW THAT THE DNA LIGASES
WILL CURE SO IT INCORPORATES THE
NUCLEOTIDINGS SUCH AS EGT AND
CNA EXAMPLE YOU SEE HERE.
IT CREATES A PERFECT SUBSTRATE
FOR BOTH LIGASES WITH THE 3
PRIME ENDS FOR EFFICIENT SEALING
EMPLOY HOWEVER WE KNOW THAT
UNDER HIGH STRESS CONDITIONS,
[INDISCERNIBLE] POLBETTA CAN
INCORPORATE THE MISMATCH, FOR
THE OXIDIZED FORM OF IGTP A OR
[INDISCERNIBLE].
SIMILARLY IF THE POLBETTA IS
ASSOCIATIONED AND ASSOCIATED
MUSEUM TAIGS AS I MENTIONED
BEFORE, IT HAS BEEN SHOWED THAT
THE PROTEIN IS A REDUCED
STABILITY AND/OR LACK OF GAP
BUILDING AND THE QUESTIONS
WHETHER THAT REPAIR IS COMING
FROM THE POLBETTA WHERE IT CAN
BE REACHED TO THE NEXT STEP IN
LIGATION [INDISCERNIBLE] OR
LIGASE 3 ALPHA.
THAT'S WHERE WE HYPOTHESIZE THAT
THE FAILURE IN THE DNA LIGASE
MALFUNCTION SENDING THE PRODUCT
CHANNELING IT DOWN THE STREAM
FOR THE PATHWAY IT COULD BE
SOURCE OF [INDISCERNIBLE]
INSTABILITY.
SO THIS IS PARTICULARLY FROM OUR
STUDIES EMERGING THAT THE
[INDISCERNIBLE] COULD CONTRIBUTE
TO THE GENOMIC STABILITY TO
NORMAL COORDINATION BETWEEN POL
BETA AND LIGASES AND
[INDISCERNIBLE].
FOR EXAMPLE, IF THE POLBETTA
CANNOT REMOVE THE DIT GROUP
MOAGHT MAY ATTEMPT TO LIGATE
[INDISCERNIBLE] ON THE P1 SIDE
AND THE FORMATION OF THE
REPAIRED [INDISCERNIBLE] WITH
THE AN [INDISCERNIBLE] DIP GROUP
HERE AND ANOTHER EXAMPLE,
EXAMPLE INCORPORATION OF
OXIDIZED GTPA, BY POLBETTA
COMPRISES BOTH, AND RESULTS IN
THE FORMATION, NOT OF ANOTHER
[INDISCERNIBLE] BUT TO REPAIR
INTERMEDIATE AND THEN 3 PRIME
AND SORT OF ATOXIC GNP.
I'VE SHOWN THERE IS SOME DETH
PROCESSING ENZYME, CANNED WHAT,
AND FAMILIES 1 CAN REMOVE THAT
LIGATION FOR THE PRODUCTS FROM
BOTH REPAIR.
AND THIS IS JUST THAT THE
DEFECTIVE SUBSTRATE PRODUCT
CHANNELING FROM POL BETA TO THE
DID, NA LIGASE CAN LEAD TO THE
INTERACTIONS IN THE BR PATHWAY
ACCORD NATION AND RESULTING IN
THE EVENTS AND DNA INTERMEDUOUS
EVER SINCE THIS, WE CONTRIBUTE
TO FIND A KEY MOLECULAR
DETERMINANT THAT INSURE THE
COORDINATION OR RESULTS IN THE
HANDLE FROM POL-BETA TO DNA
LIGASE.
FOR EXAMPLE, WE HAVE SHOWN THAT
HOW THE IDENTICAL MISMATCH
INCORPORATED BY POL BETA COULD
ASHING THAT EFFICIENT OF THE
FINAL STEPS HERE.
OUR FINDINGS SUGGEST THAT THE
PRODUCT MATCH [INDISCERNIBLE]
BUT HOWEVER, FOR THE PARTICULAR
MISMATCH, THE GTP INSERTION
POSITION C, NOW MISMATCH
INSERTION ARE COMPATIBLE WITH
THE PROCESS LEADING TO THE
LIAISON GAGE AFTER POLBETTA GT
AND CANNED WHAT CONSORTIUM
PRODUCTS.
[INDISCERNIBLE] ALL OTHER
MISMATCH WE RECEIVE THE DATP C,
THAT INSERTION AND THAT PRODUCT
CANNOT BE CHANNELED TO THE NEXT
LIGATION SET, WHICH COULD
PROVIDE AN OPPORTUNITY FOR
OPPORTUNITY PROCESSING ENZYME
AND COME IN TO PROOF READ THAT
AND MISMATCH AS WE'VE SHOWN FOR
P1 AND BEAUTIFULLY SHOWN FOR THE
STRUCTURE FOR THEM LIFE AND IT
WAS THAT GO BACK TO THE PAINT
AND GO BACK TO THE SITES FOR AN
ATTEMPT FOR POL BETA
INCORPORATION.
AND WE ALSO DEMONSTRATE THAT THE
LIGASE IS COMPROMISED BY
[INDISCERNIBLE] IN ALL POSSIBLE
CANNONICLE BASE PAIRS AND IT'S
IN THE INTERMEDIATE LIGASE AND
THE LIGATION AND DNA SUBSTRATE
WITH THE 3 PRIME [INDISCERNIBLE]
AND ALL THE 3 PRIME INSERTED
CORRECT THE BEST PART AND
[INDISCERNIBLE] AND THE MISMATCH
AND FOR EXAMPLE, GT AND AC, YOU
SEE HERE.
AND THE RESULTS SHOW THAT THE
[INDISCERNIBLE] CAN BE EFFICIENT
LIGATED WHERE WE WERE ABLE TO
SEE THE SAME EFFICIENCY FOR THE
PARTICULAR SUBSTRATE INCLUDING
AC MUCH.
AND ACTUALLY THIS HAS BEEN
SIMILARLY THE OBSERVED DISTINCT
ABILITY OF THE LIGASE 1 VERSUS
LIGASE 3 ALPHA TO LIKE THE
NUCLEAR REPAIR IN TIME WITH THE
PREINSERTED MISMATCH DEPENDING
ON THE CHEMICAL CHARACTERISTIC
OF ALL 12 POSSIBLE NONCANNONICLE
BEST PAIRS BETWEEN THE PRIMARY
TERMINALS AND THE TEMPLATE BASE,
THEY ARE--THEY MIMIC THE
BIOLOGICALLY MYCK SUBSTRATE
BEING BE FORMED DUE TO THE
MISMATCHED [INDISCERNIBLE]
INCORPORATIONS.
AND OUR STRUCTURES APPLIED AND
ALSO REVEALED THAT 1 ACTIVE
PROCESS WE DETERMINED WITH THE
MISMATCH DISTINCTLY, WE WERE
ABLE TO SO THAT THE LIAISON
ANYPLACE CAN ACCOMMODATE THE
MISMATCH IN A CONFIRMATION
DURING STEP 2 AFTER LIGATION
REACTION AND THEN
[INDISCERNIBLE] NICK DNA, AND
THEY ALSO CAPTURE THE LIGASE 1
AT THE SAME STEPS AS STEP WITH
THE NICK DNA CONTAINING THE
CANNED WHAT.
HOWEVER, WE FOUND THAT THE
ACTIVE SITES STATES ARE
VENTILATED DURING THE INITIAL
STEP 1 OF THE LIGATION REACTION,
WHILE ENGAGING WITH THE NICK DNA
CONTAINING [INDISCERNIBLE], AND
RECEIVED THE AMP, AND THE
RESIDUE, WITH THE FIRST LIGATION
STEP.
AND THEN THIS STUDY CAN FORWARD
LIGASE [INDISCERNIBLE] FOR
LIGASE 1, THAT FAVORS TO GT OR
DETER THE AC AND MISMATCH, THE
LIGATION OF THE BASIS
SUBSTITUTION ERROR INCORPORATED
AND INCORPORATED BY THE DNA
POLYMERASES, AND WE ALSO TEST
THE LIGATIONENTIOUS FICIENT
STUFF, FOR THE SUBSTRATE WITH
THE INSERTED 3 PRIMER
[INDISCERNIBLE] SIDES OUR
RESULTS DEMONSTRATE THE SEALING
OF THAT TO RECEIVE THE 3--FOR
TREATING THE 3 PRIME RIBOAT,
RIBOTC AND RIBOGC, WHICH WAS
COMPARABLE WITH THE NORMAL
COUNTER FOR THE [INDISCERNIBLE]
NUCLEOTIDES.
IS THIS ACTUALLY FOR ALL OTHER
12S MISMATCH 20 FOR RIBOGA AND
DEMONSTRATING THE
[INDISCERNIBLE] DISCRIMINATION
AGAINST RIBONUCLEOTIDES
TREATMENT TO THE REPAIR, THAT
COULD [INDISCERNIBLE] INSERTION
PRODUCTS AT THE DNA POLYMERASES
AND YOU ALSO SOLD STRUCTURAL
[INDISCERNIBLE] DNA COMPLEX,
CONTAINING THAT SIMILAR
RIBO[INDISCERNIBLE].
AND HERE TO SEE THE STRUCTURE
FOR RIBOAT TO CAPTURE THE ACTIVE
SITE AT THE LAST 3 WHICH IS THE
LAST LIGATION STEP OF--LAST STEP
OF LIGATION REACTION, AND 3
PRIME AND 5 PRIME AND RESPOND.
IN STEP 3 STRUCTURE, SHOWS AT
THE AMPs COMPLEX SUGGIESTING
THAT THE AMPs RELEASE, AND
THIS IS SUGGESTING THAT
[INDISCERNIBLE] FROM BETWEEN THE
3 PRIME AND 5 PRIME DNA FOR THIS
TO CONTINUE.
AND I THINK THAT'S ALL FOR
TODAY, I WOULD LIKE TO TURN TO
MY MEMBER [INDISCERNIBLE] AND
ALSO THE FUNDING INTERNAL AND
EXTERNAL FUNDING.
HAPPY TO TAKE QUESTIONS.
NTHANK YOU.
>> LET ME READ THE FIRST
QUESTION I SEE HERE, IT'S FROM
[INDISCERNIBLE].
HAVE YOU BEEN ABLE TO SEE IF
KNOWN B-DNA STRUCTURES FORM IN
THE PROCESS AND IF THEY'RE
INVOLVED IN ANY WAY OF THE NICK
IN.
>> NO, WE HAVE NOT.
WE HAVE NOT SEEN THAT COME FROM
THE STRUCTURES.
>> SO WHILE PEOPLE ARE THINKING
OF ARK DITIONAL QUESTIONS,
PERHAPS I COULD ASK 1.
SO YOU MENTIONED THERE ARE THIS
CANCERS YOU CAN HAVE VARIANTS OF
POL-BETA, RIGHT IN SO ARE
THOSE--ARE THOSE VARIANTS
POL BETA MORE MUTE O GENIC AND
IS THERE A CORRELATION BETWEEN
THE PARTICULAR MUTATION
[INDISCERNIBLE] IN THE ENZYME
AND ANY PARTICULAR TYPE OF
MISMATCH THAT CAN HAPPEN IN.
>> RIGHT.
SO THE PARAMETERS IS A
PREFERENCE BETWEEN A PARTICULAR
TYPE OF MISMATCH HAS BEEN SHOWN
BEFORE, I DON'T REMEMBER THAT
PARTICULAR [INDISCERNIBLE] IN MY
MIND SO IT'S REALLY A LOT BUT
SOME OF THE [INDISCERNIBLE] IS A
PREFERENCE TO INSERT A
PARTICULAR TYPE OF MISMATCH, FOR
SOME REASON [INDISCERNIBLE] DOES
NOT DO GAP FILLING AT ALL, FOR
SOME REASON, CANNED WHAT IS SOME
OTHER FORM OF THE
[INDISCERNIBLE].
NTHANK YOU.
I SEE A QUESTION FROM DEAN ROTH
WHAT DETERMINES THE LIGASE 1 OR
3 ALPHA?
>> WHAT DETERPS THE CHOICE?
>> SO SO WHAT DETERMINES THE
CHOICE.
>> THEY'RE EXCHANGEABLE.
THERE ARE 2, THAT IS WHAT I
INTRODUCED TODAY AND THERE'S
ANOTHER SUBTYPE THAT SHOWS A
LONG PATCH [INDISCERNIBLE].
STUDIES HAVE BEEN SHOWN THAT
LIGASE 1 INVOLVING MOSTLY THE
LONG [INDISCERNIBLE] SO INCLUDES
OTHER REPAIR PROTEINS LIKE PCA
AND REPLICATED POLYMERASES,
LIGASE HISTORY, AND LIGASE AND
FINAL ICING THE SHORT PATH AND
THE VR, BUT EXCHANGEABILITY AND
THEN IN CASE OF DEFICIENCY IN
LIGASE 1, LIGASE 3 ALPHA CAN
COMPLEMENT THE NICK SEALING
[INDISCERNIBLE].
>> GREAT.
THANK YOU SO MUCH.
I THINK IN THE INTEREST OF TIME.
WE WILL MOVE ON AND I WILL HAND
OVER TO PATTY.
>> HI, IT'S GREAT TO SEE
EVERYBODY, I HAVE A SPECIAL
GUEST WITH ME THIS YEAR, HE'S
VISITING US AND HE GETS TO SEE
THE TALKS TODAY AS WELL BECAUSE
NORMALLY HE'S--IT'S IN THE
MIDDLE OF THE NIGHT FOR HIM.
SO WELCOME ORLANDO.
>> HI, EVERYBODY.
>> SO ON MY DISTRIBUTION, IT'S
MY GREAT PLEASURE TO INTRODUCE
DR. ELISE, [INDISCERNIBLE], SHE
RECEIVED HER Ph.D. WHERE SHE
BEGAN HER ADVENTURES IN THE
PARTNERSHIP 1 ENZYMES AND FROM
THERE SHE MOVED TO UNIVERSITY OF
PITTSBURGH AND THEN I WAS
FORTUNATE TO RECRUIT HER TO MY
LAB WHERE SHE DID A SECOND POST
DOCK.
SHE WAS INKRE TINSIBLELY
SUCCESSFUL, AND EARNED A K99 AND
THEN WENT TO THOMAS JEFFERSON
UNIVERSITY WHERE SHE STARTED HER
LAB AND SHE WAS AWARDED AN R35,
SO SHE HIT THE GROUND RUNNING
AND THEN WE WERE VERY FORTUNATE
TO BE ABLE TO RECRUIT HER BACK
TO THE UNIVERSITY OF PITTSBURGH
WHERE SHE IS AN ASSISTA ANT
PROCESSOR IN THE DEPARTMENT OF
PHARMAICOLOGY AND CHEMICAL
BIOLOGY.
SO I WILL KEEP MY INTRODUCTION
SHORT SO YOU CAN HEAR MORE FROM
ELISE.
TAKE IT AWAY.
>> THANK YOU PATTY.
THANK YOU EVERYONE.
THANK YOU FOR HAVING ME TODAY.
SO I WILL TALK TODAY ABOUT A
PROJECT WE HAVE PUBLISHED THIS
YEAR QUITE RECENTLY THAT AIM TO
DECIDE FURTHER DYNAM
NAMES--NAMESSICS OF PARTNERSHIP
PARP1
BINDING R-LOOPS.
--AND THEY HAVE--THEY ARE
FAVORED BY NEGATIVE
[INDISCERNIBLE] AND ALSO GCQs
AND THEY ARE PHYSIOLOGY CALLED
ROLLS IN THE CELLS, BUT
UNFORTUNATELY THEY CAN BE
SIGNIFICANT FOR GENOMIC
INSTABILITY.
THEREFORE THERE ARE MANY
MECHANISMS THAT THE CELLS
EVOLVED TO EITHER PREVENT THE
FORMATION OR RESOLVING THEM
EMPLOY AND SO WHEN WE TOOK UPON
THIS PROGEC IN 2020, WE ASKED
THE REQUESTY, COULD PARP1 BE 1
OF THESE FACTORS AND WE HAD SOME
PREMISEIS FOR THAT, IN THE
RETURN, IT HAD BEEN ASSOCIATED
TO THE CANNED WHAT INTERACTOME
AND BEEN FOUND TO IRPT ACT WITH
THE DNA, RNA LEGALLIC CASE, AND
THEN LATER ON WHEN WE WERE ON
UNDERTAKING THIS PROJECT,
ANOTHER EVIDENT CAME FROM
LITERATURE SHOWING THAT PARP1
INTERACTS AND COAALATE WITH BP1
AND [INDISCERNIBLE] TREATMENT
AND DURING THE VISION OF HER
PAPER IN [INDISCERNIBLE] WE WERE
VERY HAPPY TO SEE THAT ACTUALLY
ANOTHER TEAM SHOWED THAT THE
[INDISCERNIBLE] IS SYNTHESIZED
BY PARP1.
SO THIS REASSURED US THIS OUR
HYPOTHESIS THAT PARP1 CAN DO IT
BY OWN.
ALSO BECAUSE PARP1 NOT ONLY
BINDENTIOUS FICIENTLY TO DNA
BREAKS ABOUT YOU ALSO TO
SECONDARY STRUCTURES AND IT SHOW
INTEREST THIS KNOWING MORE ABOUT
THAT, WE HAVE PUBLISHED A REVIW
IN JMB SO I INVITE TO YOU READ
MORE ABOUT THAT.
SO PARP1 BINDING DURING THOSE
STRUCTURES DIRECTLY ACTIVATE
PARP1.
SO THE QUESTION WAS COULD PARP1
DIRECTLY RECOGNIZE THE STRUCTURE
AND BE ACTIVITIED TO THE BINDING
STRUCTURES.
SO THIS IS A PAPER WE JUST
PUBLISHED IN YEN --JANUARY OF
THIS YEAR.
WE COMBINED BIOLOGY AND
MICROSCOPY AND I WILL GO OVER
THE DATA.
SO 1 THING THAT WE DID IS THAT
WE USE A PLAZ MIDS MIDTHAT WAS
GENERATED IN THE LAB AND KINDLY
PROVIED TO US BY [INDISCERNIBLE]
WHICH IS OF COURSE THE PFCAMAN.
IT'S A MOUSE GENE THAT IS PRONE
TO [INDISCERNIBLE] MUTATION, IT
WILL GO THROUGH INVITRO
TRANSCRIPTION, IT FORMS AN--IT
FORMS RNA AND IT FORMS THIS--AS
WE CAN VERIFY BY GEL HERE WE CAN
SEE AFTER THE TRANSCRIPTION,
THIS SHIFT IN THE MIGRATION,
HERE YOU CAN SEE THAT IF WE
DIGEST WITH RNA, WE CAN GET RID
OF THE FREE RNA BUT NOT THE
SHIFT, BUT THEN IF WE DIGEST,
WHICH DIGEST THE RNA IN THE
HYBRID OF THE RNA- DNA.
THE SHIFT IS ABOLERBED HERE AND
GOES BACK TO REGULAR PLAZ MIDS
MIDMIGRATION.
SO WE USE THIS SPECIMEN TO FIRST
INTERROGATE THE BINDING OF PARP1
BY FM AND HERE WE TEAMED UP WITH
THE COLLABORATOR [INDISCERNIBLE]
AT NCSU WHO HAVE THIS AFM, SO
THEY USE THIS PLAZ MIDS MIDTHAT
WE HAVE BUT HERE WE DIGEST THE
PLAZ MIDS MIDWITH APL1 THAT
GENERATES FRAGMENT IN HAD WE
KNOW THAT THERE ARE FORMS AROUND
40% FORM THE CLOSEST DMA END
WHICH WE CAN VERIFY HERE, BY
AFM, AND YOU CAN SEE HERE THAT
BY COLLECTING AFM HEIGHTS THAT
THIS LOOP IS AROUND 40% FROM THE
CLOSEST DNA.
WE THEN ENCUE BAITED PARP1 WITH
THE SAME FRAGMENT BUT WITHOUT
TRANSCRIPTION AND AS EXPECTED
PARP1 CAN BIND AT DNA ENDS,
WHICH IS QUITE HIGH BUT ALSO
HERE ON THE FRAGMENT HERE BUT
WHEN WE INCUBATE PARP1 WITH THE
LOOP AFTER RVT, CAN YOU SEE
THERE IS A SHIFT OF PARTNERSHIP
BINDING AROUND 40% OF PROCESS
UPON BINDING TO THE EXTREMITIES
WHICH SHOWS THAT PARP1 INDEED
BINDS TO ALL STRUCTURES HERE.
SO, WE ALSO WANTED TO CHECK IN
CELLS IF PRARP 1 COULD BIND IN
OUR LOOP FORMING SIDES, SO FOR
THAT WE ESTABLISH A CELL LINE IN
WHICH WE CAN OVEREXPRESS USING
DOCKS O PSYCHE LYNN, EITHER THE
WILD-TYPE OR THE MUTANT OVER
EXPRESSING THE MUTANT LEADS TO
INCREASE OF R-LOOP.
SO WE USE OUR CELL LINE HERE TO
INTERROGATE THE BINDING OF PARP1
ON THE SITES BY THE LIGATION
ASSAY USING THE [INDISCERNIBLE]
WHICH IS THE DNA, RNA HYBRID,
AND USING PARP1 ANTIBODY, HERE
ARE THE ANTIBODY CONTROLS, FOR
THE EXPERIMENT, WE NEED TO
PRETREAT OURSELVES WITH RNA-1
AND 3, WHICH GETS RID OF THE
SINGLE STRANDED ASK DOUBLE
STRANDED RNA WHICH HAS SOME
AFFINITY SO THAT MAKES OUR
EXPERIMENT MORE SPECIFIC.
AND YOU CAN SEE HERE THAT IF WE
USE THE CELL LINE EXPRESSING THE
WILD-TYPE, WE HOWEVER, YOU CAN
SEE HERE THAT'S QUANTIFIED HERE,
AN INCREASE OF FOCI FORM BETWEEN
PARP1 AND INDEX EABT BODY, WHICH
SHOWS THAT PARP1 IS ASSOCIATED
TO THE ALLUMFORMING SITES.
AND WE VALIDATED THIS IN A CELL
THAT WAS DEPLETED IN RNA
[INDISCERNIBLE] AND ALSO ANOTHER
FACTOR, RESOLUTION FACTOR.
SO OF COURSE, OUR NEXT QUESTION
WAS IS PARP1 ACTIVITY TRIGGERED
UPON BINDING.
SO TO DO THAT, WE DID THE PARP1
OR 2 MODIFICATION ASSAY INVITRO,
SO FOR THAT WE INCUBATE PARP1
WITH THE CO FACTOR NAD, WITH DMA
THAT HAS SINGLE STRAND BREAKS
WHICH IS POSITIVE CONTROL WITH
THE PLAZ MIDS MID, WITH IT TIME
REMAINS CIRCULAR TO AVOID THE
PRESENCE OF DNA THAT WOULD
ACTIVATE FOR PARP1 AND THEN WITH
THE PLAZ MIDS MIDTHAT HAVE THE
LOOP, AND YOU CAN SEE HERE THAT
UPON INCUBATION OF PARP1 AND THE
SUBSTRATE THAT WE HAVE HIGH
SYNTHESIS OF [INDISCERNIBLE] IN
POSITIVE CONTROL, A LITTLE BIT
IN THE PLASMID THAT DOESN'T HAE
THE LOOP, WHICH MAY HAVE SOME
STRUCTURES OR SOME NICKS, BUT
IMPORTANTLY WE SEE A SIGNIFICANT
INCREASE OF [INDISCERNIBLE] WHEN
WE INCUBATE WITH THE PLASMID
THAT CARRIES THE R-LOOP.
SO WE ALSO VALIDATED THAT BY IF
IN OUR RETURNED CELL LINE WHERE
WE SHOW AN INCREASE OF
[INDISCERNIBLE].
ALSO WE WERE CURIOUS TO SEE HOW
PARP1 LOOKS BY CURATED FM, SO
HERE WE HAVE TO CHANGE OUR
PARAMETERS TO SLOW DOWN THE
REACTION, AGAIN, TALK LATER
ABOUT THE REASON WHY, BUT
IMPORTANTLY WE SOLD THAT WE HAVE
PATHWAY 1, WE HAVE BRANCH, THAT
HAS--THAT IS HOW ABOUT A 40%
FROM THE CLOSEST DNA, WHICH
SHOWS IN THE PARAALATED TOP 1 IN
THE R-LOOPS.
SO HERE SO FAR WE FIND THIS
BINDING IN THE LOOP AND THE
ACTIVITY, BUT WE WANT TO
UNDERSTAND THE IMPACT OF PARP1
DELETION AND INHIBITION ON
[INDISCERNIBLE] GENOME
STABILITY.
ONE THING THAT WE DID, IS DNA
HYBRID BLAD BLOT IN OUR CELL
LINE THAT WE KNOCKED OUT IN
WHICH WE KNOCKED OUT PARP1 BUT
WE ALSO REEXPRESSED PARP1 TO SEE
IF IT RESCUES THE PHENOTYPE.
YOU CAN SEE THE ACCUMULATION AND
YOU CAN SEE THE BLOOD USING THE
ANTIBODY WITH A SIGNIFICANT
INCREASE OF RLOOP LEVEL HERE IN
THE PARP1 CELLS WHICH IS RESCUED
WHEN WE OVEREXPRESS THE PARP1
WILD-TYPE WHICH SHOWS THAT IF WE
REMOVE PARP1, THEN THERE IS
ACCUMULATION OF DNA HYBRID, DMA
RNA HYBRID IN CELLS.
OF COURSE WE ALSO REPEATED THAT
WE STOP 1 SiRNA AS WELL.
FINALLY, WHAT'S THE IMPACT OF
IPT GREATER HIB THORSITION, TOP
1 INHIBITION, ALSO LED TO AN
INCREASE OF X96 STAINING IN OUR
CELL LINE THAT ALSO THAT
EXPRESSES THE WILD-TYPE RNAH1.
YOU CAN SEE THE INCREASE.
THAT'S QUANTIFIED HERE AND
THAT'S ABOLERBED WHEN WE
PRETREAT THE CELLS WITH RNSH, TO
FIX THE CELLS IT SHOW THIS IS
SPECIFIC FORM DURING THE HYBRID
ACCUMULATION AND FINALLY WE LOOK
THEA GENOMIC INSTABILITY UPON
PARP1 INHIBITION BY FOLLOWING
THE FORMATION, WE DID THAT IN
BOTH CELLS, IN S-PHASE CELLS AND
NONS-PHASE CELLS TO SEE IF WE
HAD IPT GREATER STABILITY ONLY
LINKED TO REPLICATION STRESS.
AND WHAT WE SAW IS THAT PARP1
INHIBITION OF COURSE AS EXPECTED
LEADS TO AN INCREASE OF GAMMA H2
X IN BOTH CELL TYPES.
AND WE SAW THAT ACCUMULATION OF
R-LOOPS USING DOCKSY PSYCHE LINE
ALSO LEADING TO ACCUMULATION OF
R-LOOPS HERE BUT ONLY IN S-PHASE
CELLS WHICH IS EXPECTED BECAUSE
OF THE COLLISION BETWEEN THE
R-LOOP AND THE REPLICATION
MACHINERY.
WHAT WAS INTERESTING IS THAT WE
SHOW WHEN WE COMBINE DON,
XYPSYCHE LINE AND INHIBITOR WE
OBSERVE AN INCREASE IN BOTH
S-PHASE AND NONS-PHASE CELLS,
WHICH SHOWS ACTUALLY IT IS NOT
ONLY CHARACTERIZED IN S-PHASE
CELLS WHEN YOU INHIBIT PATHWAY.
SO PARP1 HAS ENDED THE ROLE IN
OUR [INDISCERNIBLE] IN
PRESERVING GENOMIC INSTABILITY
OF A DNA HYBRID AND WE OBTAINED
THAT SIMILARLY IN HELIX CELLS,
SO THIS IS A QUICK SUMMARY OF
THE PAPER THAT'S BEEN PUBLISHED.
I INVITE YOU TO READ OVER FOR
MORE DETAILS BUT OF COURSE NOW
WE HAVE, I WANTED TO TALK A
LITTLE BIT ABOUT OUR NEXT STEPS.
AND WE HAVE MAYBE 3 OF THOSE
QUESTIONS, SO WHAT ARE THE
DOMAINS OF PARP1 THAT ARE
INVOLVED IN THE BINDING, WHERE
IT BINDING BECAUSE IT'S RATHER
LARGE STRUCTURE AND TO DO WHAT?
AND SO, WE ARE NOW RACING THIS
FIRST QUESTION ABOUT THE DOMAINS
INVOLVED, SO THERE ARE A LOT OF
WORK, IN THE P A RPFIELD THAT
SHOWS THAT PARP1 BINDING ON THE
CANNONICLE SUBSTRATE ON THE
SINGLE AND DOUBLE STRAND BREAKS
THAT DEMON TRAIT THAT THOSE
DOMAINS, THE AND THE DOUBLE GR
BYPASSED ON THE BREAK AND THE
REMODELING OF THESE DOMAIN WILL
ALLOW FOR THE REMODELING OF THE
CATALYTIC DOMAIN AND PARP1
ACTIVATION.
SO SINCE WE SEE PARP1 ACTIVATION
ON OUR LOOP AND STRUCTURE, WE
WONDERED IF SOME OF THOSE
DOMAINS ARE ALSO INVOLVED, BUT
FIRST TO DO THAT, WE
COLLABORATED, WE ARE
COLLABORATING WITH BEN
[INDISCERNIBLE] LAB HERE AT
HILLMAN.
AND WITH THE HELP OF MATT SHAY
AND [INDISCERNIBLE] IN HIS LAB,
WE HAVE FINALLY COME UP WITH A
SITUATION ON THE
[INDISCERNIBLE].
AND SO WE ARE FOLLOWING A
PROTOCOL.
AND IT'S A 5 CHAMBER FLOW CELL
SO WE CAN FLOW THE COMPLEMENT,
SO FIRST WE FLOW THE
[INDISCERNIBLE].
AND THEN WE FLOW THE SUBSTRATE,
WE FLOW BUFFER AND THEN WE FLOW
OUR CELL EXTRACT THAT CONTAINS
THE FLUORESCENTLY TAGGED PROTEIN
OF INTEREST.
AND THEN WE CAN FOLLOW THE
BINDING ON OUR SUBSTRATE THAT
TRANSLATE INTO THIS CHEMO GRAPH
HERE POSITION HERE AND IN TIME.
SO WHAT WE DID AND WHAT THEY
DID, IS THAT SHE USED THE GENE
THAT SHE HAD IN THE PLAZ MIDS
MIDWHICH ALLOW FOR DIGESTION
WITH BBS 1 AND THE BINDING OF
THOSE [INDISCERNIBLE] HANDLE
THAT ALLOWS R-LOOP SUBSTRATE TO
BE BOUND BETWEEN THE BEADS OF
THE SIT REP AND WHAT ALSO--WHAT
THE NATALY DID HERE IS SHE DID
INVITRO TRANSCRIPTION USING ATP
WHICH ALLOWS US TO VISUALIZE THE
LOOP DIRECTLY ON THE MICROSCOPE.
SO I'M JUST GOING OVER SOME
SMALL CAMERA GRAPH WE OBTAINED
WITH THE WILD-TYPE ENZYME.
Y SOPHISTICATED YOU CAN SEE
PARP1 BYPASSEDS OR IT IS VERY
STABLE OR CAN BE VERY MOBILE
ALONG THE D, INA, AND WE CAME UP
WITH CUMULATIVE [INDISCERNIBLE]
DISTRIBUTED HERE AND THE HALF
LIFE AROUND 5.2-SECOND WHICH IS
VERY CLOSE TO THE LIFETIME
OBSERVED ON THE NICK DNA.
SO WE ARE NOW ADDRESSING THE
IMPACT OF SOME MUTATION, SOME
PARP1 MUTANTS, OLDER MUTANT WE
ARE NOT TESTING SO STAY TUNED
FOR WHAT WILL HAPPEN WITH THOSE
MUTANT AND SO THIS IS TIME FOR
ME TO THANK MY LAB, AND NATALY,
MY, Ph.D. STUDENT WHO LED TO
PROG EXPECT GRADUATED LAST WEEK
AND OUR COLLABORATORS OF COURSE,
THE LAB FOR THE [INDISCERNIBLE]
WE ARE LEADING RIGHT NOW.
AND OF COURSE, THE FUNDING AND
THANK YOU FOR YOUR ATTENTION.
>> THANK YOU.
YOUR FIRST QUESTION IS ROB ASKS
IF THERE IS A FORMATION FOR
PROCESSING AND ROLE PROCESSING?
AND THE SECOND QUESTION IS ABOUT
PARTNERSHIP 2?
>> SO ABOUT P A RP2, IT WOULD BE
INTERESTING WHEN WE KNOW WHAT
DOMAIN ARE INVOLVED WE MAY BE
ABLE TO ALSO TEST P A RP2, BUT
ALSO SEE THAT P A RP2 IS
INVOLVED THERE BECAUSE AS YOU
KNOW THE MODEL IS BIEBDING TO
THE [INDISCERNIBLE] IS VERY
DIFFERENT BECAUSE IT DOESN'T
HAVE THE SAME DNA BINDING
DOMAIN, BUT THIS IS SOMETHING WE
WILL LIKE TO TEST FOR SURE.
IT SEEMS INTERESTING.
AND THEN THE SECOND QUESTION, IS
THE [INDISCERNIBLE]OT END OF
C-TERMINUS?
I THINK IT'S ON THE END TERMINUS
IN I REMEMBER, YEAH.
>> SO I HAVE A QUICK QUESTION
EMPLOY I DON'T SEE OTHERS IN THE
CHAT YET.
DO YOU THINK THAT--SO I LIKED
YOUR QUESTION ABOUT HOW IS PARP1
DOING THIS, DO YOU THINK THAT
THE PROCESS OF THIS MIGHT BE
ENOUGH TO DISLODGE THE R-LOOP OR
DO YOU THINK THAT PARTNERSHIP IS
ACTUALLY RECRUITING SPECIFIC
HELIC CASE OR SEVERAL FOR THIS
IN.
>> THE HIGH THROUGH PUT IS THE
FIRST ACTIVITY OF PARP1 ALLOWED
FACTORS THAT ARE LESS ABUNDANT
COMPARED TO PARP1 IN THE CELL TO
QUICKLY GET THE R-LOOP
BASICALLY.
LIKE PARP1 WOULD DO ON THE
BREAK, SO HAD IS WHAT WE'RE
INTERROGATING WITH OUR
[INDISCERNIBLE] THAT HOPEFULLY
WILL BE FUNDED SOON.
[LAUGHTER]
>> DPRAIT.
AND 1 QUESTION FROM BRETT
[INDISCERNIBLE] AND THIS WILL
HAVE TO BE THE LAST 1, SO WE
HAVE TIME FOR THE THIRD TALK,
GREAT TALK, DID YOUR SUBSTRATES
FOR THE C-TRAP HAVE THE RNA
COMPONENT FOR THE R-LOOP, I MAY
ARE MISSED THIS.
>> YEAH, SO WE DO IVT BEFORE TO
TETHER THE SUBSTRATE ON TO THE
[INDISCERNIBLE], SO THE TAIL OF
THE RNA IS DIGESTED WE JUST HAVE
THE DNA HIBRIDE AND THE
DISPLACED SINGLE STRANDED DNA.
>> GREAT.
RIGHT ON TIME.
THANK YOU SO MUCH ELISE.
NTHANK YOU EMPLOY.
>> OKAY, GREAT DO I JUST JUMP?
S.
>> SO NOW WE HAVE CLAUDIA WITH
PENN STATE DEPARTMENT OF
MOLECULAR BIOLOGY.
SHE FINISHED HER Ph.D. IN THE
INSTITUTE OF BI ONY CHEMISTRY
AND WEPT ON TO POST DOCTORAL
POSITIONS AT HARVARD SCHOOL OF
DEPTAL MEDICINE AND THEN PENN
STATE WAS PROMOTED THROUGH THE
RAIRCHGS AT PENN STATE TO
RESEARCH ASSOCIATE AND ASSISTANT
PROFESSOR RESEARCH TRACK AND NOW
OF COURSE ON THE TENURED TRACK
AND HER WORK IS CURRENTLY FUNDED
BY AN NCI RO-1 FOCUSED ON PARP10
WITH REGARD TO REPLICATION
STRESS AND THAT'S GOING TO BE
THE FOCUS OF OUR TALK TODAY.
REALLY EXCITED TO HEAR YOUR WORK
CLAUDIA, THANK YOU.
I THINK YOU'RE ON MUTE.
>> CAN YOU HEAR ME NOW IN NYES
NGOOD AFTERNOON EVERYONE, THE
QUESTION IN OUR WILL BE IS TO
RECOGNIZE OW DNA IS DIAGRAMMED
AND PROCESSED.
--WHERE IT BINDS DNA POLYMERASES
AND IT PROCESSES DNA TO THE
REPLICATION REACTION EMPLOY
NONAPOPTOTIC YOU DNA LESIONS
SUCH AS THOSE INDUCED BIER UV
CAN BLOCK THE DNA POLYMERASE, SO
THIS CAN RESIDE IN DNA BREAKS
AND TRANSLOCATION, SO THE CELLS
ARE DEVELOPING FOR STAR
MECHANISM TO BYPASS THIS LESION.
IMPORTANT FOR OUR STUDY HAS BEEN
SHOWN THAT PC NA CAN BE POST
TRANSLATIONALLY MODIFIED FOR
WILL STALLS CAN GET
UBIQUITINNATEDDA THE LIGASE 164,
UBIQUITINNATED POLYMERASES ARE
ABLE TO BYPASS DNA LESIONS BUT
THEY CAN INTRODUCE MUTATIONS
ACROSS THE LESIONS.
SO WE WANTED TO FIND NEW FACTORS
THAT ARE INVOLVED IN THIS
PROCESS SO WE STARTED LOOKING
FOR PROTEINS THAT INTERACTED
WITH UBIQUITINNATED PUNISHING
CNA.
SO THIS IS HOW WE ENDED UP
STUDYING PAR10.
SO WHAT IS PAR10?
IT'S A MEMBER OF THE P A RP
FAMILY, AND AND THE SEQUENCE
STEPS, IN A MORE ADP ARRIVALS,
IN THE TRANSFER AND CHAINS THAT
CAN BE LINEAR OR BRANCHED.
THERE ARE 17--EVERYBODY TALKS
ABOUT PARP1 AND 2 BUT THERE ARE
17 MEMBERS IN THIS FAMILY, IT'S
TRUE THAT 1 AND 2 ARE THE MOST
STUDIED 1 ASK IT SHOWS THAT P A
RPs PLAY A ROLL IN SINGLE
STRADDED BREAKS AND FOR
OUR--IMPORTANT ROLE, THIS SUBSET
OF MEMBERS, THEY CAN ONLY
CATALYZE THE TRANSFER OF 1 ADP
RIEB OAZ, ON ACCEPTOR PROTEINS
THIS PROCESS IS CALLED
[INDISCERNIBLE] DPLI COSALATION
AND THIS SUBSET OF PROTEINS ARE
CALLED MONOATP RIBOSILL TRANSFER
ACES, SO P A RP10 IS A MEMBER OF
THIS SUBSET AND YOU CAN SEE IT
HERE.
NOW, WE DECIDED TO INVESTIGATE
ROLE OF P A RP10 IN DNA REPAIR
ABOUT YU P A R10, IT'S UNIQUE
WITHIN THE FAMILY BECAUSE IT HAS
A [INDISCERNIBLE] BOX
INTERACTING PEPTIDE AND WE START
AT FIRST WITH CONFIRMING THE
INTERACTION BETWEEN THE PC NA
AND PARP10 AND WE NEXT WANTED TO
KNOW WHAT'S THE ROLE OF THIS
INTERACTION IN AND SPECIFICALLY
IF IT'S ACTUALLY PARP10 REQUIRED
FOR REPLICATION RESTART THROUGH
TLS.
WE COULD SO THAT WHEN WE KNOCK
DOWN PARP10 WE SEE REDUCED PC NA
REDUCED UBIQUITINNATION, AND WE
USE SUBTLS TO SAY, AND WE SHOW
THAT YOU CAN USE THE MUTE
GENESIS IS USED AND KNOCKED
DOWN.
NOW THIS COULD BE RESTORE BIDE
REBRUCING WILD-TYPE PARP10 BUT
NOT MUTANT PARP10 THAT LACKS THE
INTERACTION WITH PC NA OR A
CATALYTIC ACTIVE MUTATION.
SO HOW DOES PARP10 PROMOTE TLS
IN WE THINK THAT BY REDUCING
UBIQUITINNATION, PARP10 IS
REDUCING THE POLYMERASES AND
WHAT WE SHOW, WE SHOW REDUCED
RAF 1 WHEN WE KNOCK DOWN PARP10
EMPLOY ACTUALLY CONFIRMING OUR
HYPOTHESIS.
SO WE HAVE THIS MODEL THAT
PARP10 IS RECRUITED TO
UBIQUITINNATED PC NA AND THROUGH
THIS CATALYTIC FUNCTION, FOR
MOST OF THE START OF THE
REPLICATION [INDISCERNIBLE]
THROUGH TLS, THIS DATA HAS BEEN
PUBLISHED SOME TIME AGO AND I
WILL SHOW NEW DATA FROMMURE ON
LAB.
FOR SYNTHESIS, FORKS CAN BE
RESTARTED BOO I RIPRIMING AND
IT'S CATALYZED BY POL, BUT THIS
LEAVES BEHIND SINGLE STRAND DID,
NA GAPS.
THESE GAPS ARE TO BE FILLED AT
THE LATER STAGE.
NOW, IN THE LAST MAYBE 2 OR 3
YEARS, YEAH, THIS SINGLE STRAND
DNA GAPS ARE VERY, VERY
IMPORTANT BECAUSE IT HAS BEEN
SHOWN THAT THE ACCUMULATION OF
HE'S GAPS ACTUALLY CORRELATE
WITH SENSITIVITY OF CANCER CELLS
TO CHEMO THERAPY, PACIFICALLY IN
THE BRACKA DEFICIENT BACKGROUND.
SO HAS BEEN SHOWN THE LOSS OF
BRACCA PROTEINS CAUSES INCREASE
IN THE SINGLE STRAND FORMATION
THIS GAPS PERSIST AND THEY CAUSE
GENOMIC INABILITY AND CELL DEATH
AND WE CAN ACTUALLY EXPLOIT THAT
VULNERABILITY FOR THERAPY.
I SKIPPED 1 SLIDE.
YES, SO RECENT STUDY HAVE SHOWN
SEVERAL MECHANISMS FOR FILLING
UP THE SINGLE STRAND DNETWORKA
GAPS.
AND IN BRACCA DEFICIENT
BACKGROUND, THE SINGLE STRAND
DNA GAPS ARE FILLED OR REPAIRED
BY A TRANSLATION SYNTHESIS, AND
1 EXAMPLE IS [INDISCERNIBLE]
THAT HAS BEEN PUBLISH EXCLUDE
HAS BEEN SHOWN ALSO THAT
INHIBITION OF TLS PROTEINS IN
THE CELLS, CAUSED A ACCUMULATION
OF THE SINGLE STRAND GAPS.
NOW BECAUSE WE SHOWED PREVIOUSLY
THAT PARP10 PROMOTES
UBIQUITINNATION OF PC NA AND THE
PC NA UBIQUITINNATION HAS BEEN
SHOWN TO PLAY A ROLE IN GAP
FILLING WE WANT TO KNOW IF
PARP10 PLAYS A ROLE IN
GENERATING SINGLE STRAND DNA
GAPS SO FOR THIS WE USE BRD
COMMENT.
WE HAVE THE CELLS WITH THE BRDU,
WE COLLECT THEM, WE EMBED THEM,
WE PUT THEM ON SLIDES, WE RUN
WITH THE SINGLE CELL AND THEN WE
TAKE PICTURES OF THE BRDU
SIGNAL, SO WE CAN SHOW, AND WHEN
WE KNOCK DOWN PARP10, WE SEE
INCREASED SINGLE STRAND GAP
FORMATION, WHEN WE HAVE THE GAP
CONDITION, 0.4 MILLI MOLAR, THIS
HAS BEEN PREVIOUS LYE SHOWN IN
THE BRCCA BACKGROUND TO GENERATE
SINGLE STRAND DNA GAPS.
NOT EVERYBODY IN THE FIELD IS
USING THIS METHOD SO WE ACTUALLY
CONFIRMED THIS PHENOTYPE WITH S1
NUCLEACE DMA FIBER ASSAY, SO IN
THIS ASSAYS WE LABEL THE CELLS,
WE BIND THE ANALOGUES, WE
PROCESS THIS, TO OBTAIN LONG DNA
FIBERS THAT WE COME WITH A
SPECIAL MACHINE AND THEN WE
LABEL WITH SPECIFIC ANTIBODIES
FOR [INDISCERNIBLE], HALF THE
SAMPLES WE TREAT WITH THE S1
NUCLEACE, SO IF THE SINGLE
STRANDS ARE PRESCRIBINGENT AS 1
WOULD BELIEVE, THEN HAVE YOU A
SHORTER SECOND TRACK, A
[INDISCERNIBLE] TRACK EMPLOY SO
WHAT WE SEE IS WHEN WE KNOCK
DOWN PARP10, WE HAVE DECREASED
RATIO IN THE [INDISCERNIBLE]
DROP, WHEN WE TREAT WITH HU, SO
THIS IS A GAP INDUCED CONDITION.
BRACCA 2 KO WE USE THEM AS A
POSITIVE CONTROL.
WE ALSO GENERATED PARP10 AND WE
CAN CONFIRM THE SAME PHENOTYPE
IN THE PARP10 KNOCK OUT.
SO, WE CAN ACTUALLY SHOW THAT
PARP10 SUPPRESSES SINGLE STRAND
DNA GAP FORMATION, UPON
REPLICATION AND STRESS.
NEXT WE WANTED TO KNOW IF PARP10
IS ACTUALLY IN THE GAP FILLING
PROCESS, SO WE USE SERVE ASSAY,
THIS IS LIKE A [INDISCERNIBLE]
ASSAY, WE USE PARP10 ANTIBODY TO
SEE IF PARP10 LOCALIZES TO
NASCENT DNA UNDER THIS GAP
INDUCING CONDITIONS, SO WHAT WE
SEE IS WHEN WE TREAT CELLS, WE
ARE RECRUITMENT OF PARP10 TO THE
SINGLE STRAND DNA GAPS.
IT HAS BEEN SHOWN TO BE THE MAIN
UBIQUITIN LIGASE FOR PUNISHING
KRRK NA AND WE WANT TO KNOW FOR
18 AND PARP10 CO LOCALIZE IN
THESE CONDITION ANDS INDEED WE
SEE INCREASE FOCI 8 HOURS SIGNAL
WHEN WE CREATE EACH
[INDISCERNIBLE].
MORE IMPORTANT WHEN WE KNOCK
DOWN PARP10, WE HAVE A REDUCED
RECRUITMENT OF RAD 18 TO IN
NASCENT TREND GAPS SO THIS
ACTUALLY SUGGIESTS THAT PARP10
WAS RECRUITING RAD 18 TO THIS
SITE.
NEXT WE WANTED TO LOOK AT THE PC
NA UBIQUITINNATION LAB AND WE
USE THE UBIQUITINNATED PC NA
THAT IS NOW AVAILABLE AND WORKS
VERY GOOD SO WHEN WE KNOCK DOWN
PARP10, WE SEE REDUCED PC NA
UBIQUITINNATION, UNDER AND WE
ALSO RESERVE FOR 1 RECRUITMENT
WHEN WE KNOCK DOWN PARP10 IN
THIS 1 LOCATION, SO I SHOWED YOU
A LOT OF FIGURES BUT THE MESSAGE
IS, WE THINK PARP10 IS PROMOTING
MOTING
RECRUITMENT OF RAD 18 TO THESE
GAPS WHERE IT PROMOTES PC NA
UBIQUITINNATION AND RECRUITMENT
OF THE POLYMERASES.
WE GENERATEDINATENTS FOR PARP10
AND WE HAVE A CATALYTIC MUTANT
AND A BIG BOX MUTANT AND WE
ACTUALLY SHOWED THAT PARP10
REQUIRES THIS CATALYTIC ACTIVITY
BUT ALSO THE BCNA INTERACTION IN
ORDER TO DO GAP SUPPRESSION.
NOW AS HAS BEEN PREVIOUSLY SHOWN
THAT THE CELLS ACTUALLY RELY ON
TLS TO FIX THIS SINGLE STRAND
DNA GAPS SO WE WANTED TO KNOW IF
PARP10 PLAYS A ROLE IN THIS, SO
WHEN WE KNOCK DOWN PARP10 IN THE
BRARCA DEFESHT BACKGROUND, WE
SEE INCREED SINGLE STRAND GAP
FORMATION WHEN WE TREAT WITH HU
OR SIS PLATIN IS OUR GAP
INDUCING CONDITIONS.
WE ALSO ARE A PARP10 INHIBITOR
AVAILABLE.
PARP1 INHIBITORS DON'T WORK ON
PARP10 EMPLOY SO WELL IS PARP10
INHIBITOR AVAILABLE ASK WHEN WE
TREAT BRACCA DEFICIENT SIZE WITH
PARP10 INHIBITOR, WE ALSO SEE AN
INCREASE IN THE SINGLE STRAND
DNA GAPS, UNDER SOME INDUCING
CONDITIONS.
NOW, VERY IMPORTANT WHEN WE HAVE
BRACCA DEPLETED CELLS AND WE
TREAT THEM JUST ABOUT PARP10
INHIBITOR, WITHOUT ANY EXOGENOUS
DAMAGE, WE SEE REDUCED VIABILITY
IN BRACCA CA AND THIS SUGGESTS
THAT THIS MAY ENHANCE THE
SENSITIVITY OF BECOME RAKRRK CA
DEFICIENT CELLS SOPHISTICATED
GENO TOXIC AGENTS SO THIS IS
SOMETHING WE WANT TO INVESTIGATE
IN THE FUTURE.
BECAUSE I ONLY HAD ABOUT 15
MINUTES TODAY AND I DIDN'T HAVE
TIME TO SHOW YOU EVERYTHING, I
JUST WANTED TO GIVE YOU AN
OVERVIEW OF WHAT WE ARE DOING
RIGHT NOW, I'M JUST GOING TO END
BY PROPOSING THIS MODEL, WE
THINK THAT PARP10, IT'S
PROMOTING GAP SUPPRESSION.
WE THINK THAT PARP10 DEPLETIONS
IMPAIRS THE ABILITY OF CELLS TO
REPAIR THIS GAPS USING
TRANSLATION SIPGHT SIS IN A
BRACCA PROFICIENT.
IF YOU HAVE BRACCA, PROFICIENT
CELLS, THESE CELLS ARE USING
HOMOLOGY BASE REPAIR TO REPAIR
THESE GAPS, BUT IN A BRACCA
DEFESHT BACKGROUND, THIS REPAIR
IS IMPAIRED, SO FURTHER LOSS OF
PARP10 REDUCES TRANSITION
SYNTHESIS GAP FILLING EMPLOY SO
THIS IS MY TAKE HOME MESSAGE FOR
YOU.
WE ARE STILL WORKING ON THIS.
I ONLY SHOW YOU PART OF THE
DATA.
WE HAVE THIS IN DIFFERENT CELL
LINES, WE CONFIRM IN DIFFERENT
SETTINGS, AND I'M GOING TO SAY
THANK YOU.
I WANT TO THANK A SPECIAL GROUP
OF PEOPLE THAT I WORK WITH,
YOU'D, ARBNAAND EMILY, AMAZING
PEOPLE AND [INDISCERNIBLE], TOO.
THIS IS OUR FUNDING FROM NETWORK
CI, AND I CAN TAKE QUESTIONS.
>> OKAY, CLAUDIA THAT WAS REALLY
FANTASTIC.
WE ALREADY HAVE A BUNCH AS YOU
CAN IMAGINE OF QUESTIONS HERE IN
THE CHAT.
THE FIRST 2 FROM OUR PREVIOUS
SPEAKER ELISE, I WILL READ THEM
OFF 1 AT A TIME.
IN OUR COMMENT IN KOAMING
ASSAYS, HAVE YOU TESTED
REEXPRESSION OF A CATALYTIC
MUTANT OF PARP10?
>> YES, I THINK WE SHOWED THAT.
YEAH, SO, WHEN WE REEXPRESS THE
CATALYTIC MUTANT, WE ACTUALLY
HAVE MORE GAPS, SO IF YOU PUT
BACK PARP10--
>> SORRY, I MISSED IT THANK YOU.
>> AND THEN THE SECOND QUESTION
IS ALSO FROM ELISE, HOW IT
PARP10 ACTIVATED, IS IT UPON
BINDING TO PC NA OR BIENING TO
DNA IN WHAT DO WE KNOW ABOUT IT?
NYES, WE ARE STILL LOOKING AT
THIS.
WE ARE STILL LOOKING AT THIS.
SO ACTUALLY PARP10 HAS AN
RRN DOMAIN, WE ARE LOOKING TO
SEE IF THIS RNN DOMAIN IS
IMPORTANT FOR BINDING OF PARP10
TO DNA, THIS IS OUR EXPERIMENTS
TO BE DONE IN THE FUTURE BUTOOSE
A VERY GOOD QUESTION.
NVERY INTERESTING, THANK YOU.
COMPREHEND NTHANK YOU SO MUCH.
>> ANOTHER QUESTION HERE ALSO
FROM A FORMER PATTY CANNED
CANNED--[INDISCERNIBLE] LAB
MEMBER SO IS RAD 18 CORRELATED
OR KNOWN TO BIND TO PAR OR
MARIN.
>> SO IT'S NOT PARALATION, IT'S
MARALATION, SO WHEN YOU ADD IT'S
CALLED MONORIBOADP, SO WE HAVE
INVITRO ASSAYS WHERE WE DO
MODULATION, WE HAVE PRELIMINARY
DATA SHOWING THAT PARP10
MODULATES RAD 18, WE ARE TILL
CONFIRMING THAT ASSAYS, NOW THEY
DEVELOP NEW ANTIBODIES TO DETECT
MONOADPRICE O ISOLATION, WE ARE
TRYING THEM IN THE LAB, IT'S A
BIT OF TROUBLE TROUBLE SHOOTING
BUT THE DAILY BASIS THEA WAS NOT
READY FOR THIS PRESENTATION BUT
THANK YOU FOR THE QUESTION.
>> WE HAVE NO MORE QUESTIONS
THERE IN THE CHAT BUT WE CAN
OPEN IT UP TO FURTHER QUESTIONS.
I HAVE A LOT OF QUESTIONS BUT
I'M GOING TO TALK TO YOU
AFTERWARDS.
>> PERFECT.
THANK YOU.
>> EVERYONE HAS TIGHT SCHEDULES,
EVERYBODY WANTS TO BE DONE BY
1:30.
>> WE CAN KEEP GOING.
>> I HAVE MUCH MORE DETAILED
DISCUSSIONS TO HAVE AND BOTHER
EVERYBODY ELSE BUT I THOUGHT IT
WAS REALLY FANTASTIC.
YOUR REVIEW THAT YOU WROTE FOR
CANCER WAS REALLY WONDERFUL AND
IT IS REALLY GREAT TO SEE THIS
MOVING FORWARD.
>> THANK YOU SO MUCH FOR
INVITING ME TO SPEAK TODAY.
N.
>> OKAY, IF THERE AREN'T ANY
FURTHER QUESTIONS, I THINK IT
JUST LEAVES US TO THANK ALL THE
PRESENTERS TODAY FOR THE
EXCELLENT TALKS AND FOR KEEPING
ON TIME.
I'M PRETTY IMPRESSED AND ALSO TO
OUR ADVISORY BOARD MEMBERS FOR
THE COORDINATION OF THIS
WEBINAR.
WE WILL SEE YOU ALL NEXT MONTH
FOR A TALK BY ELEN LEEMAN,
ENTITLED TRANSLATION SYNTHESIS,
A DNA REVIEW AND
[INDISCERNIBLE].
WE HOPE TO SEE YOU ALL THEN AND
WITH THAT I WILL SAY GOODBYE TO
YOU ALL NTHANKS EVERYBODY.
GOOD TO SEE YOU.
THANK YOU.
THANKS EVERYONE.
THANK YOU.