>>WELCOME EVERYBODY TO
THE DNA REPAIR INTEREST GROUP.
WE ARE NOW IN OUR WEBEX FROM
HOME.
IN PRESENTATIONS AND WE'RE VERY
GLAD YOU ARE ABLE TO JOIN US.
ROB SOBOL IS OUR SPEAKER
TODAY, HE WAS BORN IN NEW YORK,
BUT THEN HE WENT TO COLLEGE AT
ALGENEY COLLEGE AND THEN
GOT HIS Ph.D. AND HE STARTED
WORKING WITH SAM WILSON AT THAT
TIME WHO WAS AT NIH AND AFTER HE
GRADUATED HE WENT AROUND THE
WORLD.
HE WAS IN FRANCE AND THEN IN
GERMANY AND THEN IN NORTH
CAROLINA AT THE UNIVERSITY OF
NORTH CAROLINA CHAPEL HILL AND
HE WAS STAYING WITH SAM WILSON
BETWEEN GALVESTON TEXAS AND THEN
NIEHS UNTIL HE WENT UP TO
PITTSBURGH FOR A WHILE.
HE WAS THERE AND SINCE 2014 HE'S
BEEN IN MOBILE ALABAMA.
SX HEAT USA, THE UNIVERSITY OF
SOUTH ALABAMA, HE WILL BE
TALKING ABOUT THE MECHANISM OF
BASE EXCISION REPAIR IN HUMAN
CELLS AND WE WILL SEE IF
DR. BORE HAS ANYTHING TO ADD.
>> NOT REALLY.
I JUST WELCOME YOU ROB, I'M
LOOKING FORWARD TO THIS
DISCUSSION, THE MECHANISM, AND
EVERYTHING DONE WITH PAUL BETA
AND PROTEIN AND [INDISCERNIBLE]
AND THEN YOU HAVE ALSO
CONTRIBUTED TO MANY ASPECTS OF
BER INCLUDING AT THE NAD
METABOLISM, MEETINGS AND
DELIGHTED TO HAVE YOU SPEAK HERE
FROM FROM OUR USA--FROM USA TO
THE INTERNATIONAL FORUM.
SO WELCOME, AND THANKS AND JUST
1 MORE TIME WE HAVE A FEW PEOPLE
HERE, WE HAVE ABOUT 5 OR 6
PEOPLE WHO HAVE NOT MUTED THEIR
MICROPHONE, IF YOU KINDLY WILL
DO THAT, IT'S APPRECIATED
BECAUSE WE--OTHERWISE WE GET
BACKGROUND NOISE AND THEN,
AGAIN, QUESTIONS BY CHAT,
PLEASE.
SO PLEASE ROB.
>> OKAY, THANKS SO MUCH KEN AND
WILL AND EVERYONE ATTENDING
TODAY: WELCOME TO ALL OF YOU
FROM HERE ON THE GULF COAST AND
DISCUSS ONGOING WORK HERE ON
MECHANISM OF BASE EXCISION
REPAIR REGULATION AND THOSE OF
YOU THAT GET TO VISIT US HERE
THIS IS THE MITCHELL CANCER
EN--STRATEGIESITUTE AS PART OF
THE UNIVERSITY OF SOUTH ALABAMA
SCHOOL OF MEDICINE AND MY LAB IS
LOCATED HERE IN THE TOP AND
WE'VE BEEN VERY HAPPY IN TERMS
OF HOW WE'RE MOVING FORWARD
HERE.
I WILL GIVE YOU SOME BACKGROUND
IN TERMS OF WHERE--HOW WE GOT TO
WHERE WE ARE TODAY AND THEN I'LL
SHARE SOME WORK THAT WE'RE JUST
FINISHING UP.
SO BECAUSE I DON'T WANT TO
FORGET ANYTHING, THIS IS THE
GROUP, SO THESE ARE THE FOLKS
THAT ARE DOING ALL ACTUALLY ALL
THE PIPETTING AND EXOTIC WORK
THAT I'LL DESCRIBE TODAY AND I
WILL BE REFERRING TO THE
INDIVIDUALS THAT HAVE CONSIDERED
THROUGHOUT.
AND THEN OF COURSE I WANT TO
THANK A FEW KEY FOLKS.
THESE ARE MEMBERS OF 1 OF THE
PROGRAMS AT OUR CENTERS SO
NATALIE [INDISCERNIBLE] WHO HAS
INTRODUCED US TO CONFOCAL
MICROSCOPY AND HOW THAT CAN BE
USED TO LOOK AT DNA REPAIR
MECHANISM AT LEAST IN TERMS OF
COMPLEX ASSEMBLY, SO WHICH IS
WHAT I WILL FOCUS ON TODAY.
MARIE [INDISCERNIBLE] WHO IS A
PROFESSOR NOW IN THE DEPARTMENT
HAS A HISTORY OF WORKING WITH
VITAMINS AND N. A. D. MOLECULES
AND [INDISCERNIBLE] WHO WE'VE
WORKED ON SEVERAL PROJECTS AND
THOSE OF YOU WHO KNOW SHE'S A
STRUCTURAL BIOLOGIST AND WE'RE
ALL HAPPY THEY ARE PART OF OUR
CANCER CENTER AND OUR
UNIVESITY.
SO, AS WILL ALLUDED WE'VE BEEN
WORKING ON BASIC CISION REPAIR
AND FOR A LONG TIME AND IN
PARTICULAR, WE'VE ALWAYS BEEN
THINKING ABOUT IT FROM THE
PERSPECTIVE OF PORP, AND TO GIVE
YOU BACKGROUND, BUT JUST TO
REORIENT EVERYONE, THESE PLAY A
CRITICAL ROLE IN GENOME
STABILITY AND THESE PROTEINS ARE
ALL TO SOME EXTENT SOMEWHAT
AFFECTED IN CANCER IN VARIOUS
WAYS, AN INTERESTING ASPECT FOR
EXAMPLE IS [INDISCERNIBLE] CROSS
LINK REPAIR, WITH NEW FINDINGS
AND THEN MANY OF THESE FACTORS
AS I LIKE TO CALL THEM IN BASIC
[INDISCERNIBLE] ARE CONSIDERED
NOW TO BE NOVEL AND DRUGGABLE
TARGETS FOR SYNTHETIC LETHALITY
AND IT RAISES THE QUESTION OF
WHAT OTHER FACTORS CAN WE THINK
ABOUT AND WHAT I ALSO LIKE TO
THINK ABOUT, WELL, 1 THING TO
POINT OUT HERE IS OF COURSE THAT
PART 1 AND ININCREASE IN
BODYITORS ARE NOW CONSIDERED AS
EFFECTIVE SMALL MOLECULES AND
TREATMENT OF CANCER IN
PARTICULAR FOR CANCERS WITH
DEFECTS IN BRCCA 1 AND 2, WE ALL
KNOW THESE FINDINGS BUT THIS
SPEAKS TO WHAT WE'RE ENDEAVORING
TOO IS UNCOVER THESE NOVEL
EXCISION REPAIR OR SINGLE STRAND
BREAK ARE REPAIR AND THOSE
FACTORS THAT CAN BE REGULATED
AND TARGETING SYSTEMYETED FOR
EFFECTIVE RESPONSE IN CANCER, SO
THAT'S THE BIG PICTURE OF OUR
OVERALL THEME IN THE LAB.
AGAIN GOING BACK IN TIME AS WE
THINK ABOUT EXCISION REPAIR
ORIGINALLY 1 THING ABOUT IT IS A
VERY SIMPLE FORMAT, BASE DAMAGE
IS INN DUCED BY A VARIETY OF
SOURCES MOST OF THE CASES THAT
DAMAGE IS REMOVED BY GLUE
MARIOUS COSALATE AND BY BASIC
SITES AND POLYMER ACE DATA
CARRIES OUT 2 OF THESE TYPICAL
FUNCTIONS IN THE CONTEXT OF THIS
PATHWAY, BOTH TAYLORING THE GAP
WITH THE PRIME DRP MICE FUNCTION
AND THEN SYNTHESIZING DNA TO
FILL THE GAP AND THEN
SUBSEQUENTLY DO IT WITH THE
LIGATION STEP.
SO, WE KNOW OF COURSE THAT BER
DATA IS RESPONSIBLE FOR A LOT OF
DATA.
SO BETA CAN COMPLEMENT THE KNOCK
OUT TO PROVIDE SURVIVABILITY
WHEREAS THE LYASE DEFICIENT
MUTANT DOES NOT APPEAR TO
PROVIDE THAT COMPLEMENTATION, SO
THAT TELL US THAT THE FUNCTION
IS IMPORTANT AND NOT JUST THE
PROTEIN ITSELF OR AT LEAST IN
THE CONOF EXPOSURE LIKE AN
ALKALINE OR IN THIS CASE
[INDISCERNIBLE], WHEN WE THINK
ABOUT BASE EXCISION REPAIR
OSINGLE BREAK REPAIR, AS I
POINTED OUT WE HAVE TO REALIZE
THAT IT PLAYS A CRITICAL ROLE
AND FACTOR IN MAMMALIAN CELL
THIS IS IS A COMPLICATED ENZYME
WITH NUMEROUS DOMAINS AND THE
GENERAL CONCEPT HERE IS THAT
PARK RECOGNIZES THE BREAK AND IN
THE PRESENCE OF N. A. D.
GENERATES THESE POLYMERS OF
RIBOSE THAT FUNCTION AS A
LANDING PAD IF YOU WILL TO
FACILITATE RECRUITMENT AND THEN
OF COURSE WE HAVE TO REMEMBER
THAT THERE ARE IN EFFECT
REVERSAL REACTIONS, THE MAIN
ENZYME [INDISCERNIBLE] WHICH
REMOVES PRIMATES MARLY ALL THE
RIBOSE MOIT ESTIMATE THAD AND
THEN 3 WHICH TENDS TO BREAK DOWN
THAT POLYMER AS ITS DEGRADING.
OF COURSE, PARK 1 IS HIGHLY
REGULATED AS EVIDENCED BY A LOT
OF THE WORK AND PEOPLE LISTENING
TODAY AND THIS IS THROUGH POST
TRANSLATIONAL MODIFICATION AMONG
OTHER MECHANISMS AND WHAT WE'RE
GOING TO TOUCH ON IN THE FINAL
SECTION OF TODAY'S TALK IS SER6
AND HOW IT MAY REGULATE PARK AND
P A RP AND EXCISION REPAIR.
SOPHISTICATED AS I'VE BEEN
SAYING WE WE'VE BEEN LOOKING AT
THE QUALITY RIBOSE, WHEN YOU
HAVE A BER SUPPRESSED SITUATION
FOR EXAMPLE, AND YOU EXPOSE AN
ALKACCTINATOR AND YOU SEE THE
ACCUMULATION BUT IF YOU HAVE AN
ACTIVE OR HYPER ACT BASE
EXCISION REPAIR SITUATION WITH
LIMIT THE POLYMER ACE BET ASSUEY
THE ACCUMULATION OF THE P A RP
BETA IS FURTHER SUPPRESSED YOU
SEE IT BRANDED ON THE EXPRESSION
OF POLB, TO ELEMINATE THAT,
KIEWM AWLINGS AND THEN
INTERESTINGLY IF YOU EXPRESS THE
MUTANT THAT CANNOT REPAIR THAT
OR TRIM THE GAP, WHEN YOU SLIDE,
THAT DOESN'T SEEM TO EFFECT THE
P A RP ACTIVATION.
SO THIS SUBJESTS THAT IN THAT
MODEL THE BER LESION MIGHT BE
VERY STRONG FOR ACTIVATING A
LESION.
SO WE'VE THOUGHT ABOUT THAT IN
THAT MODEL FOR MANY YEARS AND
THEN WHAT'S INTERESTING, TOO, IS
THAT NOT ONLY IS P A RP A
CRITICAL FACTOR BUT THEN THE NAD
FACTORS INTO THIS.
SO YOU CAN SUPPRESS THIS HYPER
TENSATIVITY BECAUSE OF P A RP
ACTIVATION SIMPLY BY ADDING NAD
PRECURSORS, IN THIS CASE N MN
THAT ACTS AS A PRECURSOR TO
SYNTHESIZING N. A. B. AND NOW
THIS IS OLDER WORK AND I WILL
DISCUSS MORE DETAILED COMPONENTS
THAT REALLY POINT AT N. A. D.
BEING A CRITICAL FACTOR HERE.
SO AGAIN, WE LOOK AT MAMMALIAN
BASE REPAIR FROM THIS VIEW POINT
AND AS WE'VE DISCUSSED AND
REPORTED IN THE ABSENCE OF LET'S
SAY COMPLETION OF REPAIR, YOU
END UP WITH THIS RIBOS E
SIMULATION AND THE RESULTING
CELL DEATH.
THIS PUTS P A RP UPSTREAM OF
POLB AND NAD WOULD BE AN
ESSENTIAL CO-FACTOR AND THIS HAS
BEEN SUGGESTED AND SUPPORTED BY
MANY OTHER STUDIES THAT WHEN
DEPENDING ON NAD LEVELS YOU
MIGHT SEE VARIOUS LEVELS OF CELL
DEATH ASSOCIATED WITH THE BER
ACCUMULATION.
OF COURSE, NAD IS NOT SYNTHESIS
IS NOT SPRAYED FORWARD, IT IS
MANY DIFFERENT PATHWAYS, IT
CONTRIBUTES TO THE PRODUCTION OF
NIEE, AUDIENCE D AND THIS IS
JUST SOME OF IT LAID OUT HERE
FOR YOU ALL TO SEE AND OF COURSE
THE NAD THAT'S SYNTHESIZED IS
IMPORTANT FOR VARIOUS POST
TRANSLATIONAL MODIFICATIONS
INCLUDING B-ASSIMILATION AND
MONOCLONAL ASSIMILATION AND
POLYB ASSIMILATION AS WELL AS
METABOLIC FUNCTIONS THAT N. A.
D. CONTRIBUTES TO AND 1 CAN
EFFECT THIS AND ASK QUESTIONS
ABOUT NAD'S ROLE IN REPAIR VERY
SIMPLY BY DEPLETING THIS WITH A
NICE ININCREASE IN BODYITOR OF
THIS NAM T ENZYME--NEVER MIND,
I'M NOT POINTING--NAM T ENZYME
YOU CAN INHIBIT EFFECTIVELY WITH
FD6 ANDEE USE THIS A LOT TO
EFFECT BASE CISION REPAIRS.
THIS IS PATHWAY GIVES MOLECULE,
VERY EFFECTIVE INHIBITOR WITHIN
ABOUT 24 HOURS, GOING TO SEE 80%
PROGRESSION OF NAD.
SO WE ASKED THE SIMPLE QUESTION,
IF WE TAKE A LOOK AT THE VARIETY
OF [INDISCERNIBLE] AGENTS THAT
ALL DAMAGE THE DNA IN VARIOUS
DIFFERENT WAYS, AS CAN YOU SEE
HERE BY MEASURED BY THE CONIC
CHIP ASSAY, CAN YOU SEE ENHANCED
DNA DAMAGE IN A VARIETY OF
DIFFERENT AGENTS AND THE
QUESTION SIMPLY IS, IF WE CHANGE
THE NAD LEVELS DOES THAT AFFECT
THE ACCUMULATION OR REPAIR OF
THIS DNA DAMAGE, AND AS YOU
MIGHT PREDICT, THIS IS NOT
UNIVERSAL PHENOMENON.
SO FOR EXAMPLE, A TOPOCID HAS
NO--IS NOT AFFECTED BY LEVELS OF
NAD, SO WE SUPPRESS THE NAD
LEVELS TO ABOUT 10 OR 15% OF THE
TOTAL.
WE ASK IF A TOPOSIDE REPAIR, AND
MEDIATED DNA DAMAGE IS THERE AND
YOU CAN SEE THERE'S NO EFFECT,
YET AGENTS LIKE MMS, WHICH
HISTORICALLY IS THOUGHT TO GO
THROUGH BASIC EXCISION REPAIR,
YOU SEE THIS VERY ROBUST AND
SIGNIFICANT EFFECT ON THE DAMAGE
LEVELS AND THE REPAIR LEVELS SO
AGAIN SUPPORTING A ROLE FOR NAD
IS A CRITICAL BASE EXCISION
REPAIR FACTOR.
SO 1 OF THE QUESTIONS THAT WE
HAD IN A RECENT REPORT IS CAN WE
MODULATE REPAIR COMPLEX
FORMATION WITH ALTERATIONS IN
NAD LEVELS AND WE DID THIS IN
THE CONTEXT OF A BIGGER STUDY
AND THIS IS JUST SOME SUMMARIES
HERE, SO WE LOOKED AT THE EFFECT
OF EITHER SUPPRESSION OF NAD
THROUGH FK H6, OR
SUPPLEMENTATION OF PRECURSORS 6
HOURS IN BOTH FETAL BOVINE SERUM
AND IN HEATED ACTIVATED BOVINE
SERUM AND IN SERUM FREE MEDIA
AND REALLY WHAT WAS INTERESTING
HERE WAS THAT WHEREAS WE SEE A
ROBUST EFFECT IN THIS CONTEXT,
EITHER IN SERUM FREE MEDIA AND
IN--REALIZE THAT FETAL BOVINE
SERUM DEGRADES THE COMPOUND AND
IT DOES POINT AND REPAIR BECAUSE
OF THAT DECREASED NIEE, AUDIENCE
D LEVEL AND WE WILL GET INTO
THIS A BIT MORE IN THE MEET OF
THE STORY HERE.
SO AGAIN, AS WE THINK ABOUT DNA
REPAIR FROM A GLOBAL PERSPECTIVE
IS OBVIOUSLY MANY, MANY
DIFFERENT PATHWAYS AND PROTEINS
INVOLVED.
P A RP IS EMERGING TO PLAY A
ROLE NOT ONLY IN BASE EXCISION
REPAIR BUT NUCLEOTIDE EXCISION
REPAIR AND VARIOUS COMPONENTS OF
DOUBLE STRAND LAYER REPAIR AND
IF YOU LAYER ON TOP OF THAT, THE
NAD AND P A RP FORMATION THAT'S
NECESSARY AND ALL OF THOSE CO
FACTORS THAT ARE NECESSARY, YOU
CAN SEE ENTIRE PATH, ALL THE
PATHWAYS SEEM TO BE POST
TRANSLATIONING MODIFIED IN SUCH
A WAY THAT NAD IS PROBABLY A
VERY CRITICAL FACTOR.
THAT'S 1 OF THE COMPONENTS WE'VE
BEEN INTERESTED IN PURSUING.
WHAT WE'LL TALK ABOUT TODAY IS
IN THE CONTEXT OF BASE CISION
REPAIR SO AGAIN IF WE LOOK AT
THIS MODEL, WE ALL KNOW THE
COMPLEXITY HERE AS WE START TO
THINK ABOUT IT, ABOUT 20 ENZYMES
INVOLVED, BUT 1 OF THE THINGS
THAT THIS OBVIOUSLY CHANGES IN
THE CONTEXT OF [INDISCERNIBLE]
WORRY ABOUT AND SO IN TERMS OF
THE CHROMATIN MEDIATED REPAIR OR
THE REPAIR A CHROMATIN P A RP
BECOMES A VERY IMPORTANT PLAYER
TO HELP UNWIND THE COMPACT
SITUATION WHERE THE DNA IS
EMBEDDED IN THESE HISTONES AND
OTHER FACTORS AND THAT GIVES
ACCESS TO THE DAMAGE DNA, THIS
IS NOT A SURPRISE.
P A RP IS WELL DOCUMENTED TO
HAVE AIR ROLE IN CHROMATIN
RELAXATION AND IF YOU SCOUR THE
LITERATURE, CAN YOU SEE A HOST
OF FACTORS THAT HAVE BEEN
SUGGESTED TO BE P A RP DEPENDENT
CHROMATIN RELAXATION OR
NUCLEOSOME EVIKS FACTORS AND SO
WE NOW MORE THINK ABOUT THE VER
OR SINGLE STRAND BREAK REPAIR TO
HAVE A STEP WHERE THERE'S AN
ESSENTIAL COMPONENT HERE THAT
FACILITATE THIS AND OUR GOAL IS
TO UNDERSTAND IN THE CONTEXT OF
BASE EXCISION REPAIR WHAT ARE
THOSE FACTORS?
AND OF COURSE P A RP IS NOT ONLY
PLAYING A ROLE IN BASE EXCISION
REPAIR OR SINGLE STRAND REPAIR,
IT PLAYS AN PRNTD ROLE IN SINGLE
REPAIR, AND OF LATE IT PLAYS AN
IMPORTANT ROLE FOR THE
REPLICATION SPORTS, MAYBE WITH
REGARD TO THE [INDISCERNIBLE]
FRAGMENT ACTIVATION AND THEN
ULTIMATELY THE REGULATION OF THE
REPLICATION FOR IT.
SO IN OUR CURRENT WORK WE'RE
INTERESTED NOT ONLY IN THE TEMP
RETURN OF RESULTSLY MAPPING IF
YOU WILL THE INTERACT OHM SO WE
CAN DISCOVER THESE FACTORS THAT
ARE ESSENTIAL FOR NOT ONLY
SINGLE STRAND BREAK REPAIR AND
BASE EXCISION REPAIR AND BUT
ALSO REPLICATION FORKS AND I'M
REALLY JUST TO FOCUS ON THE BER
COMPONENT FOR TODAY'S TALK.
SO THIS IS OUR GLOBAL
PERSPECTIVE AND HOW TO MOVE
FORWARD AND UNCOVERING THESE
FACTORS.
TEMP RETURN OF RESULTSLY, WE'LL
FIRST START OFF WITH TEMPORALLY
MASKING THESE FACTORS AND WE'VE
EMBRACED A FEW OF THESE AND
THESE CLASSICAL PROTEIN INTERACT
OME ANALYSIS AND I WILL TALK ON
THAT TODAY AS WELL.
SO FOR THOSE WHO HAVE NOT USED
THIS, I KNOW MANY OF YOU
PROBABLY HAVE, BIOID IS KIND OF
A UNIQUE SYSTEM, THERE'S MANY OF
THESE SIMILAR SYSTEMS OUT THERE
NOW, BIOID ALLOWS 1 TO USE YOUR
PROTEIN WITH THIS E.COLI FACTOR
BIRA, AND IT'S A MODIFIED FACTOR
AND SO IN THE PRESENCE OF
ELEVATED BOTTLE OF BIOTIN 1 THEN
EFFECT BIOACCUMULATING THOSE
PROTEIN IN ABOUT 10 NANO METERS
SO WE THINK OF THIS AS A WAY OF
TEMPORALLY MAPPING THE BER
INTERACT OHM FOR EXAMPLE.
SO THAT THAT END WE'VE TAKEN IT
UPON OURSELVES OF LOOKING AT ALL
OF THESE PROTEINS, I WILL TOUCH
ON A FEW OF THEM TODAY SO FOR
EXAMPLE, GLUE MARIOUS COSLACES,
THE WAY WE,A PROACH THIS IS WE
KNOCK OUT THE GLUE MARIOUS
COSLACE AND WE REEXPRESS THIS
CONSTITTATIVELY OR REGULATED AS
A FUSION AND THEN THIS GIVES AN
OPPORTUNITY TO IDENTIFY FOR
EXAMPLE, DAMAGE INDUCED INTERACT
OHM OR CONSTITTATIVE INTERACT
OHM WHAT HAVE YOU.
--MPG AND THE GLUE MARIOUS
COSLACE AND THEN WE REEXPRESS IN
THE NEXT SLIDE, I'LL JUST SHOW
YOU WE REEXPRESSED THE
[INDISCERNIBLE] AS A PREFUSION
TO BIRA, AND OF COURSE WHAT
WOULD WE EXPECT IF WE DO THIS,
RIGHT?
WE KNOW THAT THE METHYL GLUE
MARIOUS COSLACED ARE ESSENTIAL
FOR THE REPAIR FOR EXAMPLE OF
THEN ALKAALATED BASIS AND
CONTEXT OF BASIC EXCISION REPAIR
AND WE WOULD EXPECT TO FIND
FACTORS THAT ARE CRITICAL HERE
AND 1 CAN'T FORGET THAT WHEN YOU
EXTO THINGS LIKE MMS, YOU END UP
ENGAGING OTHER PATHWAYS LIKE THE
[INDISCERNIBLE] FAMILY THAT ARE
ALSO RESPONSIBLE FOR REPAIRING
THESE BASES AND WE'RE PROBABLY
NOT SHOCKED TO BE ABLE TO FIND
FACTORS BECAUSE IT'S BEEN
SUGGESTED FOR EXAMPLE THAT THIS
MPG GLUE MARIOUS COSLACE MAY
INTERFERE WITH AND INTERACT WITH
THE [INDISCERNIBLE] AT THE PH2,
AND CONVERSELY IN BASE EXCISION
REPAIR IN A RECENT REPORT IT WAS
SUGGESTED THATOXIDATIVE DAMAGE
MAY ACTUALLY INCORPORATE UVDB FR
EXAMPLE AS A CENSUS, SO WE
VISIONED THAT THERE'S PROBABLY
FACTORS TO FACILITATE SOME OF
THESE PATHWAYS FOR ITS PATHWAY
COORDINATION SO WE'RE EXCITED TO
CONTINUE TO PROCEED ON THIS,
THIS IS GENERALLY DONE THE
FOLLOWING WAY.
WE DO MULTIPLE EXPOSURES AND
THEN GLUE MARIOUS COSALATE ALL
THESE PROTEINS, WE GENERALLY MAP
THIS IN CONCERT WITH A
CORRELATION.
IN OTHER WORDS WE ENVISION IF WE
EXPOSE THE CELLS DIRECTLY WE
SHOULD SEE P A R ACCUMULATION
AND THEN WE COORDINATE IT AND
MAKE SURE ALL OUR SAMPLES LOOK
LIKE THIS.
AS YOU MIGHT EXPECT, THE MPG IS
LABELED BY THE BIOTIN AS WELL AS
THE NUCLEACE, WE ONLY SEE A
SLIGHT DAMAGE-INDUCED INDUCTION
OF THE BIOTIN AALATION OF THE
CANNED WHAT.
INTERESTINGLY WE SEE HISTONE H3
AS THE LABELED THROUGHOUT
SUGGESTING THAT THIS IS ALL
OBVIOUSLY WITHIN THE CHROMATIN
REGARDLESS OF THE AMOUNT OF
DAMAGE WHICH IS AN INTERESTING
FINDING IN AND OF ITSELF AND
THEN WE PROCEED AND WE'RE REALLY
FORTUNATE TO WORK WITH
[INDISCERNIBLE] AND ZHANG AT THE
UNIVERSITY OF PITTSBURGH AND WE
GIVE THEM DOZENS AND DOZENS OF
SAMPLES AND THIS GOES THROUGH
STANDARD WORK FLOW AND NOT
SURPRISINGLY, WE HAVE IDENTIFIED
NOW OTHER FACTORS AS WELL AS
PROTEINS INVOLVED IN B REPAIR
ALTHOUGH JEN IS WORKING ON THIS
AND I'M SURE SHE'S LISTENING AND
SHE WOULD BE SHOOTING ME HERE IF
WE REVEALED WHAT THEY ARE AT THE
MOMENT BUT WE'RE--STAY TUNED,
WE'RE CONTINUING TO WORK ON
THAT.
SO TD OTHER BIG COMPONENT THAT
WE'RE PURSUING AT THE MOMENT IS
ACTIVATION IN PARTICULAR WITH
REGARDS TO SINGLE STRAND BREAK
REPAIR AND BASIC EXCISION
REPAIR, THIS IS ALSO QUITE
PRODUCTIVE AND WE HAVE 2 GROUPS
OF PROTEINS WE IDENTIFIED HERE,
YOUR TYPICAL BASIC EXCISION
REPAIR PROTEINS, PRIMARILY P A
RPs AND CC1 AS FACTORS OF
BIOACCUMULATED BY P A RP, BUT
INTERESTINGLY ENOUGH WHERE
REPLICATION FACTORS SUCH AS RFC1
AND TCNA, WE THINK THESE FALL
INTO 2 CATEGORIES, PROBABLY ON
THE LEFT, THE ROLE OF P A RP IN
BASE EXCISION REPAIR OR
CLASSICAL SINGLE STRAND BREAK
REPAIR AND PROBABLY ON THE RIGHT
THE MECHANISM OF P A RP INDUCED
OR P A RP MEDIATED REPLICATION
STRESS AND SO FOR TODAY'S TALK,
WE WILL JUST FOCUS ON BASE
EXCISION REPAIR AND SINGLE
STRAND BREAK REPAIR AS WE
CLASSICALLY REFER TO IT.
SO WHAT WE'RE INTERESTED HERE IS
UNCOVERING THOSE PROTEIN AND
HISTONE ACETYLATION AND CODE IF
YOU WILL AND REGULATES CLASSICAL
BASE EXCISION REPAIR.
SO, AS A I REFERRED TO EARLIER,
WE ENVISION THIS P A RP 1
MEDIATED CHROMATIN UNWINDING AND
SUBSEQUENT RECRUITMENT OF THE
MODIFYING COMPLEXES AND WE
CONSIDER THAT THERE'S A NUMBER
OF THINGS TO THINK ABOUT, DNA
DAMAGE RESPONSE FACTOR TO THE
HISTONE CHAPERONS, CHROMATIN
MODIFIERS AND EPIGENIC MODIFIERS
AND OUR HYPOTHESIS HERE IS THAT
THERE'S A DIN,A DAMAGE RESPONSE
AND HISTONE MODIFICATION CODE
THAT DICTATES AND REGULATES BASE
EXCISION REPAIR OR SINGLE STRANT
BREAK REPAIR,--SO HOW WE DO
TRACK THIS?
AGAIN WE'VE BEEN INTRODUCED TO
CONFOCAL MICROSCOPY SO WE CAN
TRACK COMPLEX ASSEMBLY AND
DISASSEMBLY IN THE LIVE CELL.
THE THIS INVOLVES THE LASER
INDUCED DNA DAMAGE, AND
PRIMARILY FOCUS ON EGFP TAG, POL
B AND EGFP TAG ERCC1 AND TAGOT
END TERM GNAWS AND TAG ON THE
C-TERMINUS, AND THEN WE COMBINE
THAT WITH INHIBITORS OR SELECT 2
KNOCK OUTS SO WE CAN PROBE
FUNCTION AND INVOLVEMENT IN
COMPLEX ASSEMBLY.
AND WE'VE BUILT THE SYSTEM
PRIMARILY BUILT BY JILL ANDREWS
WHO IS THE MANAGER OF OUR
MICROSCOPY UNIT WHICH HE CALLS
MIDAS AND IT GIVES US AN
OPPORTUNITY TO HAVE A REALLY
HANDS ON APPROACH TO GET THESE
PLOTS HERE IF YOU WILL, SO FOR
EXAMPLE, THE 405 LASER,
PRIMARILY MAKES SINGLE AND
DOUBLE STRAND BREAKS, OF COURSE
THERE'S A SPECTRUM OF THINGS
DEPENDING ON THE DOSE AND THEN
WE ALSO USE THE 355 NANO METER
LASER, THIS TENDS TO BE
PRIMARILY MORE BASE CISION
REPAIR AND SINGLE STRAND REPAIR
LESIONS AND CAN YOU SEE THE
KINETICS OF RECRUITMENT OF THE
DOUBLE STRAND BREAK PROTEINS AND
BASE PAIR PROTEINS ARE QUITE
DIFFERENT AND WE USE THIS TO
FOLLOW THE COMPLEX ASSEMBLY AND
DISASSEMBLY AND WE REALLY
DEVELOP 2 SYSTEMS, SOMETHING WE
CALL MID AS [INDISCERNIBLE].
WHERE OF COURSE WE HAVE CONTROL
CELLS THAT HAVE NO LASER INDUCED
DAMAGE AND WE MONITOR THAT AS A
CONTROL, THAT AREA OF INTEREST
AND THEN 10 OTHER CELLS ARE
MONITORED IN PARALLEL AND THEN
THEY'RE CONTINUING OVER TIME TO
ALLOW US TO GET THESE PLOTS,
EPPED UP DOING THIS ABOUT 4
TIMES OR MORE SO WE CAN GET AN
AVERAGE OF 40 OR MORE CELLS TO
GET A LOT OF RECRUITMENT FOR
EXAMPLE AS SHOWN HERE FOR CC1
AND THIS PARALLEL ANALYSIS AND
PROVIDES QUANTIFICATION OF
REPAIR FOR PROTEIN RECRUITMENT,
WE DO 10 CELLS PER EXPERIENT,
AT LEAST 40 FOR ANY OF THE PLOTS
I WILL BE SHOWING AND THEN WE
HAVE SOMETHING CALLED
SERO-ANALYSIS WHERE WE LOOK AT
THE SAME CELL OVER AND OVER
AGAIN AND THIS GIVES US AN
OPPORTUNITY TO MAKE SURE WE'RE
NOT MISSING ANYTHING THE FIRST
15-20 SECONDS IN THAT ANALYSIS.
AND THIS PROVIDES RAPID
SELF-QUANTIFICATION OF PROTEINS.
NOW AGAIN WE'RE LOOKING AT THE
STEPS OF BASE EXCISION REPAIR,
THERE ARE 2 CRITICAL ENZYMATIC
ROLES AND SO TO ADDRESS THIS
WE'VE SIMPLY EXPRESSED DUE TO
THE WILD TYPE OR THE 2 MUTANTS
AS EFF FUSIONS AND YOU CAN SEE,
BASICALLY WE DON'T SLEEP APNEA
AND OBESITY ANY CHANGE
SURPRISINGLY THAT IS THE PEAK
RECRUITMENT TIME AND THE HALF
LIFE IS ALL SIMILAR SUPG JESTING
SORRY, SUGGESTING THAT
RECRUITMENT IS NOT DEPENDENT ON
FUNCTION AT LEAST IN THE THE 2
OR 3 CELLS--EXCUSE ME--2 OR 3
CELL TYPES WE'VE LOOKED AT.
WE ASK ASK THE QUESTION,
HOWEVER, BECAUSE WE'RE
EXPRESSING THIS USUALLY ON LENTI
VIRUS, IS THIS AFFECTED BY THE
LEVEL OF THE ENDOGENOUS PROTEIN
AND SO HERE WE'RE JUST COMPARING
THE 3 PROTEINS OF INTEREST
EXPRESSED AS A FUSION TO THE
SAME SITUATION WHERE WE KNOCKED
OUT ENDOGENOUS LEVEL AND THESE
ARE PROTEINS ARE EXPRESSED AS
CAS 9 RESISTANT FUSIONS AND YOU
CAN SEE EFFECTIVELY NO CHANGE IN
TIME OF PEAK AND NO CHANGE IN
HALF LIFE SO REALLY THERE'S NO
CHANGE, IN OTHER WORDS THE
ENDOGENOUS PROTEIN IS NOT
AFFECTING THAT.
HOW ABOUT IF THE CHANGING LEVEL?
OF COURSE THOSE OF THAT YOU WORK
WITH ANY KIND OF LENTI VIRUS,
IT'S PROBABLY EXPRESSING THE
PROTEIN AT 10 AND 20 OR 50 TIMES
HIGHER THAN NORMAL AND SO, IN
THE FIRST TASK WE ISOLATED BY
FLOW, HIGH MEDIUM AND LOW
EXPRESSERS, THESE HAVE NO CHANGE
IN RECRUITMENT TIME OR PEAK
LIFE, PEAK TIME OR HALF LIFE, SO
IT DOESN'T SEEM TO BE A
CONCENTRATION DEPENDENT BUT THAT
STILL ALWAYS WORRIES US SO THE
GOAL WAS TO TAG THE GENES WE'RE
INTERESTED IN, AND THE END
TERMINUS, AND THE C-TERMINUS
ABOUT THIS DATA HERE IS THE END
TEMPERATUREUS AND WE THINK WE'VE
ONLY TAGGED 1 ALLELE, THIS GIVES
A VERY NICE EXPRESS WITH
PRIMARILY NUCLEAR WE'VE WE'VE
SEEN THIS EXTREMELY
[INDISCERNIBLE].
IN FACT WE SEE A HIGHER P-KITE
AND THIS IS THE RESULT OF
SIGNATURES SIGNAL TO NOISE
LEVEL, AND ENDOGENOUS IN THE
BETA BUT WE DON'T SEE ANY PEAK
RECRUITMENT OR HALF LIFE.
BUT THIS IS BECOMING 1 OF THE
MORE POWERFUL WAYS WE WOULD LIKE
TO MOVE FORWARD.
WHAT ABOUT EXOCC1.
WE THINK OF POLB AND EXOCC1 AS A
HETEROGENEOUS ROW DIMER WHETHER
THEY EXIST AS A HETEROGENEOUS
ROW DIMER HERE, IT'S HARD TO
KNOW BUT WE DO KNOW IF WE KNOCK
OUT--OOPS SORRY--IF WE KNOCK OUT
ECHO CC1, WE SEE LESS CHROMATIN
AND NUCLEAR ACCUMULATION OF POL
B POTENTIALLY BECAUSE OF ITS
INTERACT BUT ALSO BECAUSE OF THE
UBIQUITYINATION NEGOTIATED WITH
SEPARATED INTO 2 PROTEINS AND
THIS IS JUST PLOTTED RIGHT HERE.
BUT WE CAN ASK QUESTIONS WHETHER
OR NOT THE RECRUITMENT IS
DEPENDENT ON EXOCC1.
SO WE SIMPLY KNOCKED IT OUT AS
SHOWN HERE AND YOU CAN SEE AS
YOU MIGHT PREDICT IN THE ABSENCE
OF XRCC1 THERE'S NO RECRUITMENT
OF POLB TO THE SITE OF DAMAGE.
IT'S NOT ONLY THE EXPRESSION OF
XRCC1 THAT IS NEEDED, IT'S
ACTUALLY THAT INTERACTION, SO
YOU CAN MUTATE POLB SO IT CANNOT
INTERACT WITH XRCC1, AND YOU SEE
NO RECRUITMENT ON THESE
CONDITIONS, SO THE RECRUITMENT
THEN REQUIRES BOTH THE PRESENE
OF THE PROTEIN AND INTERACTION
BETWEEN THE 2 AND AGAIN, THIS
TRIP, THIS TM-MUTANT IS A MUTANT
THAT'S A SEPARATION OF FUNCTION
AND IT BLOCKS THAT INTERACTION.
SO TO BE HONEST, I STILL DON'T
KNOW IF THESE PROTEINS ARE
RECRUITED AS A HETEROGENEOUS ROW
DIMER AND THAT'S AN ONGOING
QUESTION THAT WIEWR INTERESTED
IN.
--WE'RE INTERESTED IN.
IN OTHER WORDS IS POLB THERE AND
XRCC1 COME TO THE SIDE?
AND WE DON'T KNOW THAT
SPECIFICALLY AT THIS POINT.
SO WE KNOW HOWEVER THAT BOTH
POLB AND XRCC1 IRPT ACTION AND
THE EXPRESSION IS ESSENTIAL
COMPLEX [INDISCERNIBLE]--WHAT
ABOUT DISASSEMBLY?
SO AS I SHOWED YOU HERE, WE GET
VERY NICE TREATMENT OF XRCC1?
WE DO THIS NOW IN THE ABSENCE OF
POL B, IT'S DOWN STREAM MIGHT
NOT EXPECT ANY EFFECT BUT IN
FACT, IN THE ABSENCE OF POLB, WE
SEE NO RECRUITMENT EFFECT BUT
LOSS OF POLB SHOWS US HERE THAT
XRCC1 DEMONSTRATES SLOWER
DISASSEMBLY FROM THE REPAIR
COMPLEX IN THE ABSENCE OF POLB.
SO THERE'S SORT OF A--2 SIDES OF
THIS COIN.
YOU NEED XRCC1 FOR POLB TO COME
THERE AND THEN YOU NEED POLB FOR
IT TO BE THERE FOR THE
DISASSEMBLY COME IS KIND OF
INTERESTING AND THEN WE BUILT
ANOTHER VECTOR SYSTEM WHERE WE
EXPRESS THE EGFP TAG, XRCC1
EITHER IN THE ABSENCE OF AN
EMPTY SECOND CASSETTE, OR
THE--OR THE POLB THAT'S
RESISTANT TO THE GUIDE RNA AND
WHEN YOU EXPRESS THE EMPTY
SECOND CASSETTE, AGAIN, YOU SEE
THE SAME PERSISTENT FOCI OF
XRCC1 BUT THEN THE PRESENCE OF
POLB IN THAT KNOCK OUT REVERSES
THAT PHENOTYPE AS WE SEE HERE.
SO THIS SUGGESTS TO US THAT
AGAIN, YOU HAVE TO HAVE POLB
THERE TO AFFECT THAT DISASSEMBLY
WHICH IS A NOVEL FINDING AND
THIS S&P JUST THAT SERO-ANALYSIS
AND WE ARE NOT MISSING ANY TIME
POINTS OF XRCC1 RECRUITMENT.
SO POLB AND XRCC1 ASSEMBLY
SHOULD BE REGULATED BY POLB
RIBOSE OF COURSE, THIS IS THE
MODEL WE'VE BEEN THINKING ABOUT
WHICH IS CONSIST WENT THIS UPPER
MODEL HERE AND SO AT FIRST WE
WENT AHEAD AND JUST DID SOME,
YOU KNOW IMMUNE O FLUORESCENCE
AND YOU CAN FIX YOURSELF AFTER A
MINUTE AND SEE THAT NICE POLB
FOCI, AND THEN YOU CAN STAIN
WITH AN ANTIP A R-ANTIBODY IS
SIMILAR TO WHAT EVERYONE USES
AND IT'S RIGHT ON TOP OF EACH
OTHER.
IF YOU HAVE DONE THIS, YOU
REALIZE THIS IS PAINFUL BUT BUT
WE CAN CONSISTENTLY DEMONSTRATE
THAT THE IMPORTANCE OF P A RP IN
THIS CONTEXT, IF WE KNOCK OUT P
A RP, WE SEE A SUPPRESSION OF
THAT RECRUITMENT AND CONSISTENT
WITH THE REQUIREMENT FOR THAT
POLB RIBOSE, AND SIMILARLY IF WE
INHIBIT P A RP WE SEE A COMPLETE
ATTENUATION OF THE POLYMER WITH
THE P A RP INHIBITOR, YOU SEE
THAT POL BETA STICKS AROUND AND
DOESN'T COME OFF THE SITE.
WE SEE THE SAME THING FOR XRCC1
FOR EXAMPLE.
INTERESTINGLY, [INDISCERNIBLE]
WHICH IS THE ENSIEM THAT REMOVES
THE VERY LAST ADP RIBOSE, AND
NOT SURPRISING BECAUSE IT'S 1
ADP RIBOSE UNIT.
SO AGAIN, THE MODEL THAT WE'RE
THINKING ABOUT HERE, WE USE
THESE 2 PROTEINS TO UNDERSTAND
THE COMPLEX ASSEMBLY AND
DISASSEMBLY STEP, AND WE ENHANCE
OUR MECHANISTIC ANALYSIS BY MORE
EFFECTIVELY TRACKING POLYB
RIBOSE, SO THAT IMMUNE O
FLUORESCENCE IS NOT FOWN TO DO
AND NOT REALTIME AND NOT
QUANTITATIVE AND WE ASKED THE
QUESTION--CAN WE MONITOR THE
ACCUMULATION OF P A R FROM THESE
REVIEWS EXPERIMENTS, YOU KNOW TO
GET AWAY FROM THIS VERY PAINFUL
APPROACH AND TO DO THAT, WE
DEVELOPED SOMETHING WHICH WE
CALL REAL P A R WHICH IS A P A
R BINDING DOMAIN FUSED EGFP AND
THIS ALLOWS US TO MEASURE P A R,
FORMATION AND ACCUMULATION IN
REALTIME.
AND IT TURNED OUT TO BE NOT SO
TRIVIAL IN THAT THERE ARE 10
MORE BINDING DOMAINS, WE CREATED
THIS REAGENT FOR ALL OF THEM.
THEY ALL EXPRESS REASONABLY
WELL, SOME OF THEM HAVE UNIQUE
EXPRESSION PROFILES AND WE
TESTED THEM ALL FOR 4 OR 5 AND
355 NANO METER RECRUITMENT AND
ONLY 1 OF THEM RECRUITED THE
LASER INDUCED DNA DAMAGE,
FORTUNATELY AT LEAST, AND THIS
IS SOME OF THAT DATA HERE,
HERE'S AN EXAMPLE OF THAT
RECRUITMENT, WE GET A VERY NICE
RECRUITMENT AND AGAIN IT'S A P A
R-BINDING DOMAIN LINKED TO GFP
AND SO THE CARTOON OF HOW WE
ENVISION IT WORKING AND IT'S
QUITE SPECIFIC.
SO WE GET VERY NICE RECRUITMENT
KINETICS AND XRCC1 TO SOME
EXTENT BUT YET IF WE MUTATE THE
RESIDUES KNOWN TO BLOCK P A R
BNDING YOU ELIMINATE THAT
RECRUITMENT SO WE'RE KIND OF
EXCITED ABOUT THAT AND IN
ADDITION, SIMILAR TO WHAT WE SAW
FOR POLB AND XRCC1, IF YOU
INHIBIT P A RP, YOU INHIBIT ANY
RECRUITMENT AND IF YOU INHIBIT P
A R, THE RECRUITMENT LEVEL
PERSISTS OF THIS REAL P A R
REAGENT.
SO WE'RE USING THIS NOW IN ALL
OF OUR STUDIES.
NOW WE CAN USE THIS TO PROBE P A
RP FORMATION KINETICS ALONG WITH
ASSEMBLY AND DISASSEMBLY.
SO 1 OF THE QUESTIONS WE ASKED
IS LOSS OF POLB IMPACT INDUCED P
A R FORMATION, TO REMIND YOU IN
THIS CONTEXT WE SAW THAT WHEN WE
EXPOSED THIS HIGH LEVELS OF
[INDISCERNIBLE], WE GET
ACCUMULATION OF P A RP, AND THIS
CONTAINS XRCC1 OR REAL P A R AND
THIS IS THE REAL PAR DATA.
SURPRISINGLY WE SEE NO CHANGE IN
THE ACCUMULATION OR APPEARANCE
OF THE PAR SIGNAL AS EVIDENCED
BY THE REAL PAR REAGENT.
AND THIS SUGGESTS POTENTIALLY
THERE AT THESE ADMITTEDLY VERY
LOW LEVELS OF DNA DAMAGE, IS A
BACK UP ENZYME THAT'S
FACILITATING REPAIR OF THE P A
R ACTIVATION LESION SUCH AS
POLQ, POLI, OR OTHERS, SO IS
PAR JUST SO PROBUST THAT WE
ACTUALLY DON'T SEE IT
ACCUMULATED VERY MUCH AND THAT'S
SOMETHING UNDER STUDY NOW.
AGAIN WE'RE INTERESTED IN THESE
FACTORS.
WE'VE LOOKEDDA THE THESE ON 2
PROTEINS HERE, WE LOOKED AT REAL
P A R AND THEN WHAT ABOUT
NIEE, AUDIENCE D, DOES THAT
CONTRIBUTE IN THE SAME CONTEXT
IN TERMS OF REGULATING COMPLEX
ASSEMBLY, AND AS WE REFERRED TO
EARLIER, YES, IN VERY
EFFECTIVELY, IF YOU SUPPRESS
LEVELS OF CELLULAR NAD AND THEN
ASK CAN THEE PROTEINS RECRUIT,
YOU BASICALLY DON'T SEE ANY
RECRUITMENT AND THAT IS LIKELY
BECAUSE YOU CANNOT SEE A
MEASURABLE LEVEL OF P A RP
ACTIVATION BUT WHAT ABOUT
ALTERATIONS IN NAD VIABILITY, IS
THAT SOMETHING WE REALLY HAVE TO
WORRY ABOUT?
WELL, AGAIN WE TRIED TO MAKE
WILL ARGUMENT HERE THAT WE
SHOULD CONSIDER FACTORS SUCH AS
NAD AS DER REGULATORY FACTORS,
IN FACT THE BIOSYNTHESIS OF NAD
IS WHICH IS A SUBSTRATE FOR BOTH
IS COMPARTMENTALIZED AND HIGHLY
REGULATED, THESE HAVE BEEN
LINKED TO THE AGING PROCESS AND
CANCER RELATED BIOSYNTHESIS IN
DIFFERENT TISSUES SO IT'S BECOME
VERY COMMON THESE DAYS THAT
INCREASING NAD VIABILITY MAY
PROVIDE AN OPPORTUNITY TO
INCREASE CELLULAR DNA REPAIR
CAPACITY, I WILL POINT OUT
HOWEVER, IT REMAINS TO BE
DETERMINED IF TOO MUCH OF A GOOD
THING CAN ALSO BE DETRIMENTAL,
AND THAT'S SOMETHING WE ARE ALSO
CONSIDERING.
SO IT BECAME REALLY INTERESTING
THEN, WE WERE THEN WORKING WITH
MARIE [INDISCERNIBLE], WE WERE
ABLE TO GET A NICE SOURCE OF
[INDISCERNIBLE] OR DISCIPLINARY
HYDRONIKO TIN O ROBOSIDE, IT'S A
PRECURSOR TO NAD, READILY GETS
SYNTHESIZED INTO NAD AND IN FACT
UNDER NONSTRESSED CONDITIONS, IT
CAN RAISE THE LEVEL FOR A FEW
HOURS AND OVER 10-15 FOLD HIGHER
THAN FORMAL, THIS BECOMES
INTERESTING BECAUSE IN SOME CELL
TYPES WE FIND THAT P A RP MUST
BE IF YOU WILL--IT DOES NOT HAVE
ENOUGH NAD AVAILABLE.
SO HERE'S AN EXAMPLE OF A CELL
TYPE WHERE IF WE ECPOSE THE
HYDROGEN PEROXIDE AND WE DROVE
FOR PAR ACCUMULATION, IT'S
PRETTY LOW BUT WHEN WE TREAT THE
SOLES WITH NRA WE SEE THIS
EXTREMELY ROBUST AND P A RP
ACTIVATES AND SUGGESTING THAT
IT'S MISSING A LOT OF SUBSTRATE
AVAILABILITY IN THIS CONTEXT SO
WE ASK SIMPLY IF WE TREAT WITH
NRH DOES THAT AFFECT BASE
EXCISION REPAIR AND OF COURSE
IT'S WHAT WE SEE AND WE SEE
INCREASED RECRUITMENT, INCREASED
XRCC1 RECRUITMENT AND OF COURSE
INCREASED PAR-ACCUMULATION.
SO AGAIN WE THINK THAT NAD HAS
TO BE AN IMPORTANT FACTOR WHEN
CONSIDERING ALL THESE THINGS.
BUT THESE ARE NOT THE ONLY--P A
RP IS NOT THE ONLY PLAYER IN THE
FIELD, WHAT ABOUT MODIFIERS AND
WHEN WE'RE THINKING ABOUT NAD,
OBVIOUSLY THAT BRINGS US TO THE
[INDISCERNIBLE], AND WE FOCUSED
A LITTLE TIME ON CERT 6, NOW WHY
IS CERT 6 IMPORTANT FOR BER?
I WILL GET TO THAT IN THE NEXT
SLIDE.
WE'VE DONE THAT BY KNOCKING CERT
6 OUT IN ALL OF THESE CELLS AND
OF COURSE CERT 6 WAS SUGGESTED
THAT THE ROLLING BASIS REPAIR BY
AN UNKNOWN MECHANISM SOMETIME
AGO, WE KNOW AT LEAST FROM
REPORTS THAT IT CAN MODIFY BER
PROTEINS SO WE HAVEN'T--WE
HAVEN'T ASKED IF ANY OF OUR
PROTEINS ARE MODIFIED BY SERT 6
YET, THAT'S UNDERSTUDIED, WE DO
KNOW HOWEVER THAT CERT 6 AND
KNOWN TORACETALATE HISTONS THAT
CERTAINLY CAN BE PART OF AN
INTERESTING COMPONENT WITH
REGARD TO CELLULAR BASE EXCISION
REPAIR.
AND THEN IT'S ALSO KNOWN THAT
CERT 6 CAN EFFECT CHROMATIN
RELAXATION AND THIS IS THE
MEASUREMENT OF THE CONTEXT OF
DOUBLE STRANDED BREAK FORMATION,
WE'RE NOT SURE IF THAT'S THE
CONTEXT OF BASE EXCISION REPAIR
AND MAYBE MORE RELEVANT IS THAT
CERT 6 HAS BEEN SHOWN TO
ACTIVATE P A RP 1 IN RESPONSE TO
DOUBLE STRANDED BREAKS BUT NOT
BY DEASSIMILATION BUT IN FACT BY
THE ROBUST ACETYLATION SO WE
THOUGHT ABOUT THAT AND WONDERED
CAN THAT BE RESPONSIBLE FOR WHAT
YOU KNOW--CAN THAT BE RELATED TO
WHY CERT 6 WILL EFFECT BASE
EXCISION REPAIR IN THE EARLIER
STUDIES SO WE TOOK A LOOK AT
THAT AND AGAIN USING THOSE KNOCK
OUTS I REFERRED TO AND SHOWED,
SURPRISINGLY WE GET ALMOST A
COMPLETE ATTENUATION OF POLB
RECRUITMENT AND REDUCED DNA
DAMAGE WHEN WE KNOCK OUT CERT 6,
AND SIMILARLY ALMOST A COMPLETE
ATTENUATION OF XRCC1 IN THE
ROBBERIA SENSE OF CERT 6, BUT P
A RP ACTIVATION IS NOT AFFECTED
IN FACT IT'S ALMOST THE REVERSE,
WE ALMOST SEE MORE P A RP
ACTIVATION IN THE ABSENCE OF
THIS, SO THIS CERT 6 THEN
EFFECTS THE RECRUITMENT BY
MODULATING P A RP THE CC1
COMPLEX OR EVEN THE CHROMATIN
STATE ANY TYPE OF RECRUITMENT
BUT DOES NOT IMPACT LASER
INDUCED P A RP FORMATIONS, SO
IT'S DIFFERENT THAN WHAT'S SEEN
FOR DOUBLE STRAND BREAK REPAIR
AND WE THINK THIS IS INTERESTING
AND SO THE FACTORS THEN THAT ARE
AFFECTING THIS COMPLEX ASSEMBLY
ARE SHOWN HERE, BUT OF COURSE WE
HAVE TO THINK--SO NOW WE'RE
PURSUING THIS IN TERMS OF ALL
THE OTHER FACTORS THAT ARE
NECESSARY, AS WELL AS THE OTHERS
BUT OF COURSE WE ONLY LOOKEDDA
THE A FEW OF THESE PROTEINS,
THEY'RE OBVIOUSLY MORE, WE HAVE
AN ONGOING PROJECT WITH
[INDISCERNIBLE] AND TREY
[INDISCERNIBLE] AND THEY'VE
BUILT NOW SOMETHING THEY CALL
HIGHER UP MODEL OF DNA REPAIR
WHICH IS FROM OMICS SCALE
INTERACTION DATA SO BASICALLY GO
GOES OUT AND PULLS TOGETHER ALL
KNOWN PHYSICAL INTERACTIONS, IN,
RNA, CO EXPRESSIONS, PROTEIN CO
EXPRESSIONS, GENETIC CO
EXPRESSIONS GENETIC LEARNING
TYPE OF MODEL FOR DNA REPAIR, I
WILL NOT GO THROUGH ALL THE
DETAILS, THIS IS HOPEFULLY
COMING OUT SOON, BUT AS YOU
DRILL DOWN TO DNAREPAIR
COMMENNENTS THERE ARE 4 MODELS
THAT CAN COME OUT OF THIS DATA
AND OF COURSE WE WANTED TO ASK
THE QUESTION FWE TAKE A LOOK AT
THE SHORT BATCH FACTORS NOT ONLY
DO WE HAVE TO CONSIDER DNR CC1
BUT WE HAVE TO FACTOR THOSE IN
AS WELL AND WE LOOKEDDA THE
THOSE IN ADDITION TO OUR WORK
WITH POLB AND XRCC1 AND ASKING
IF ARE THEY ALL POLB DEPENDENT
AND THEY HAVE A STRONG P A RP
DEPENDENCE.
INTERESTINGLY SO THAT LIGASE 3,
AND MULTIPLE--ALMOST ENTIRELY AS
WELL AS APDX, AND IT HAS A
PARTIAL P A RP DEPENDENCE OF
COURSE, REAL P A R, YOU DON'T
SEE ANYTHING IN THE ABSENCE OF P
A RP ACTIVATION AND THEN IF YOU
LOOK AT THOSE KINDS OF PEAKS
THERE'S INTERESTING DATA SHOWING
UP HERE AND THEY'RE CORRELATED
AND THEN HAVE YOU HALF LIFE OF
RECRUITMENT IS ALSO AN
INTERESTING DATA SET TO LOOK AT.
WHAT IT WILL ITS US HERE AS WE
LAY THIS ON TOP OF EACH OTHER
THAT SOME OF THESE PROTEIN VS
VERY SIMILAR KINETICS AND THIS
IS POLB AND APTX AND THEN EXRCC1
AND OTHERS ARE CO LOCALIZING AND
THEN OF COURSE THE REAL P A RP
PERSISTS SO WE'RE ERPSYCHESSED
TO THINK ABOUT HOW WE REORGANIZE
THIS, BUT IF WE SEE THE
RECRUITMENT THEN THE XRCC1
RECRUITMENT TENDS TO BE ALL ON
ITS OWN, PROBABLY COORDINATING
THESE 3 FACTORS HERE, AND THEN
THAT CHANGES WHAT WE LOOK AT
HERE TO MAYBE SOMETHING MORE
LIKE THIS AND OF COURSE, THEY'RE
PROBABLY NOT ALL COMING HERE
TOGETHER, WE'RE PROBABLY LOOKING
AT MULTIPLE EVENTS, IN A SINGLE
LASER INDUCED SHOT, BUT AT LEAST
THIS IS 1 WAY TO THINK ABOUT IT
AS WE MOVE FORWARD.
SO FOR ALL OF THESE FACTORS
THOUGH, HOW DID THE CERTS, P A
RP AND CHROMATIN MODIFIERS
IMPACT THIS ASSEMBLY AND THERE
ARE RULES FOR EACH?
AND THIS IS SORT OF AN ONGOING
STORY.
AND SO, I'M GOING TO END WITH
THAT MODEL THERE, AND I'LL BE
HAPPY TO TAKE QUESTIONS MOVING
FORWARD.
THANKS VERY MUCH.
>> THANK YOU FOR THE VERY NICE
TALK THERE.
WE HAVE A NUMBER OF QUESTIONS
SUBMITTED IN THE CHAT.
WILL, DO YOU WANT TO START?
>> I WILL START, CAN YOU MUTE
PLEASE PEOPLE MUTE.
WHAT NAD LEVELS AND CANCER
CELLS, ARE THEY ELEVATED?
IT'S A COMPLEX QUESTION, YOU CAN
ADDRESS IT.
BOB, YOU'RE MUTED.
>> OKAY, HOW'S THAT, BETTER?
SO THANK YOU FOR MUCH FOR THAT
QUESTION, IT'S A COMPLEX
QUESTION AND WE'VE NOT DIRECTLY
COMPARED CANCER CELLS TO
THEIR--LET'S SAY NORMAL BECAUSE
HOW CAN YOU DEFINE THAT?
SO WE DON'T KNOW THE ANSWERS,
SPECIFICALLY, I WOULD SAY IT'S
REALLY HARD TO TELL THAT
QUANTITATIVELY BECAUSE WHAT ARE
YOU COMPARING IT TO?
>> SOME CANCER CELLS ARE
DIRECTLY AFFECTED IN THE NAD
METABOLISM AND THEREFORE CLEARLY
HAVE VERYIANT LEVELS OF NAD.
>> YEAH, WE'VE SEEN A LOT OF
DIFFERENT PHENOTYPES, WE'VE SEEN
SITUATIONS WHERE LET'S SAY
PRECURSOR SUPPLEMENTATION DOES
NOT GIVE RISE TO MORE NAD
PRODUCED AND OTHERS WHERE
PRECURSOR SUPPLEMENTATION DOES
GIVE RISE TO NAD SO THAT'S ALL
THE FACTORS THAT ARE NECESSARY
FOR BIOSYNTHESIS AND HOW THEY'RE
AFFECTED.
SO I DON'T THINK IT'S AN EASY
ANSWER BUT I THINK IT'S A GREAT
QUESTION.
>> I'M GOING TO GO TO THE NEXT
PERSON HERE, BEVERLY, DO YOU
HAVE ANY THOUGHTS ABOUT POLB
RECRUITMENT, SITES EVER DAMAGED
IN THE MITOCHONDRIA HA AND
WHETHER THEY'RE P A RP
DEPENDENT?
>> IT'S I GOOD QUESTION.
I THINK IT'S WELL ESTABLISH THAD
P A RP IS NONAPOPTOTIC NOT IN
THE MITOCHONDRIA SO THAT MAY NOT
BE THE CASE.
WE HAVEN'T LOOKED AT ALL AT POLB
RECRUITMENT IN TERMS OF LATE
DAMAGE INDUCED RECRUITMENT
BECAUSE THAT'S A WHOLE OTHER SET
OF EXPERIMENTS, BUT I WOULD SAY,
IT'S LIKELY NOT P A RP INDUCED.
>> LET ME JUST SAY THAT MANY
PEOPLE INCLUDING OURSELVES FIND
THEM--THAT MAY BE VARYING IN
TYPES OF CELL TYPES AND I KNOW
IN SOME OF THE P A RP MEETINGS,
THAT'S A BIG CONTROVERSY BUT
WE'VE NOT MEASURED IT SO IT'S
HARD TO KNOW.
>> WE HAVE 1 FROM AMANDA
[INDISCERNIBLE], DO YOU SEE THE
RECRUITMENT IS FROM OTHER DNA
DAMAGE RESPONSE P A RPs,
WHETHER IT'S 2 OR 3, SEEN NO
RECRUITMENT OF POL B IN THOSE
CASES HERE?
>> RIGHT, WE HAVEN'T--THAT'S A
GREAT QUESTION AND I'M GLAD YOU
SAW THAT.
YES THERE'S DEFINITELY A
DIFFERENCE IN P A RP 1 AND KNOCK
OUT INHIBITION SINCE AT LEAST
INHIBITORS WILL P A RP 2, AND
THEY ARE PLAYING A ROLE AND WE
HAVEN'T SPECIFICALLY ASKED THAT
QUESTION, BUT THAT'S A GREAT
QUESTION.
>> SO THEN SARAH ASKED IF YOU
HAVE TRIED SUPPLEMENTATION OF
OUR CELLS WITH NAR TO INCREASE
NAD LEVELS AND YOU DID THAT BUT
MAYBE YOU COULD EXTEND ON THAT?
>> YEAH, SOPHISTICATEDY WE TRIED
A WHOLE BUNCH OF AGENTS AND NAR
WORKS REALLY WELL IN STRESSED
CELLS, WE TEND NOT TO SEE BASAL
INDUCTION OF NAD LEVELS BY NR
ALONE AT LEAST IN THE CELL TYPES
WE'VE LOOKED AT.
IT DOES WORK REALLY WELL TO
COMPLEMENT, LET'S SAY FK86
INDUCED NAD SUPPLEMENTATION AND
NR WILL BRING IT BACK TO NORMAL.
THAT'S BEEN REPORTED.
WE'VE REPORTED ON IT AND WILL
AND OTHER VS REPORTED ON THAT,
BUT THE NRH TENDS TO BE A MUCH
MORE--BECAUSE WE DON'T ACTUALLY
KNOW HOW NRH IS CONVERTED IN NAD
WE DON'T KNOW THE FACTORS THAT
ARE NECESSARY AND IT SEEMS TO BE
MUCH MORE ARE BUST MECHANISM OF
NAD BIOSYNTHESIS IF YOU WILL
STIMULATION.
>> FROM KYLE MILLER HERE TO
EVERYONE, DOES NICOTINE O MID
WHICH IS A SEROTONIN INHIBITOR
SHE SAYS TREATMENT GIVE YOU THE
SAME RESULT AS 6 KNOCK OUT OR
BER FACTOR RECRUITMENT WITH DNA
DAMAGE?
>> GREAT QUESTION.
>> --DEACETYLATION DEPENDENT?
>> THAT'S A GREAT QUESTION, WE
DON'T KNOW THE ANSWER.
>> CAN I JUST THROW IN A
QUESTION HERE, ROB, SO YOU
MENTIONED THAT SOMETIMES THAT
THE RECRUITMENT THAT IS
INDEPENDENT UPON ACTIVITY OF THE
PROTEIN OR SOMETHING, SO WE TALK
A LOT ABOUT PHENOMENON CALLED
PIGGYBACK OR SOMETHING ELSE,
THAT VERY OFTEN AND WE'VE DONE A
LOT OF RECRUITMENT EXPERIMENTS,
YOU SEE OTHER THINGS BEING
RECRUITED THAT MAY OR MAY NOT GO
THERE IN ITS OWN FUNCTION BUT
BEING CARRIED ALONG WITH THE
PROTEIN THAT BIND TO AND SINCE
WITH ALL THESE MEASURES, YOU DO,
DO YOU HAVE ANY FEEL FOR THAT
AND OF COURSE, IT'S HARD TO
DIFFERENTIATE THESE THINGS BUT
HOW DO YOU SEE THIS?
IS RECRUITMENT ALWAYS AN
INDICATOR OF AN ACTIVE FUNCTION
OF THE PROTEIN AT THE SITE OF
THE--
>> THATYA A GREAT QUESTION.
THAT'S WHY I RAISED THAT 1
STATEMENT THERE THAT WHAT WE
STILL DON'T KNOW IS THE--LET'S
SAY THIS CONTEXT POLB AND XRCC1
ARE THE ONLY HETEROGENEOUS ROW
DIMER AT SITES THAT ARE DAMAGED
AND SOME OF THE BIOLOGY WOULD
ARGUE THAT THEY HAVE INDEPENDENT
FUNCTIONS SO THEY'RE LIKELY NOT
ALWAYS A HETEROGENEOUS ROW DIMER
BUT YOU KNOW WE'RE TRYING TO
BUILD THE FLUORESCENT TOOLS AND
OTHER AND THE ANSWER OF THAT
QUESTION WE DON'T KNOW THE
ANSWER, IT'S JUST BEING BROUGHT
IN BECAUSE IT'S BOUND AS AN
EXAMPLE BUT WE DON'T KNOW THE
ANSWER.
>> YOU HAVE A QUESTION KEN?
>> YES, THERE'S 1 FROM--I HAVE A
QUESTION.
>> WHENEVER I HAVE PATIENTS WITH
SERIES POINTSA DOSA PIGMENTOSA,
THEY HAVE UDDB2, MUTATIONS THEY
CLINICALLY GET CANCER, HAVE YOU
STUDIED CELLS THAT ARE DEFICIENT
IN UDD2?
NA'S A PA, RP--WE'VE NOT, THE
PAPER WAS PRETTY EXCITING TO
DEMONSTRATE THAT IT HAD A ROLE
OR LIKELY AS A ROLE IN THE
LESION RECOGNITION INOXIDATIVE
DAMAGE, WE DID NOT ANSWER THAT
QUESTION, I THINK WE KNOCKED IT
OUT IN SOMEBODY WAS LOOKING AT
THAT YOU BUT I DON'T KNOW THE ANSWER
TO THE QUESTION,.
>> ANOTHER QUESTION CAME IN, DO
YOU FIND RAD 51 IN YOUR OGG1
BIOID EXPERIMENTS, I KNOW I
SHOULDN'T ASK BUT I DO ANYWAY
SINCE--
>> YEAH, WE DO, WE ARE JUST
ANALYZING THE DATA SO WE DON'T
KNOW IT.
>> IS THE EFFECT ON RECRUITMENT
WITH THE CERT 6 KNOCK OUT
ANYTHING TO DO WITH THE HISTONE
ACETYLATION OR CORRELATION?
MAYBE THE RIBOSOLLATION THAT IS
ACTIVATED BY DNA DAMAGE?
>> THAT'S THE QUESTION, THAT'S
WHAT WE'RE TESTING NOW.
WE'VE ACTUALLY KNOCKED OUT ALL
THE [INDISCERNIBLE] AND ASKING
GLOBALLY HOW THEY AFFECT THIS
BUT RIGHT NOW WE'RE FOCUSING
THIS STUDY ON CERT 6 AND THEN
THE QUESTION BECOMES IS THE HOST
OF ACETYLATION POTENTIAL SITES
AND WE'RE ADDRESSING THOSE
QUESTIONS AS WE SPEAK.
>> I THINK THAT'S WHAT WE HAVE
FOR QUESTIONS.
>> THERE'S A COMMENT, THERE'S A
COMMENT FROM SARAH
[INDISCERNIBLE], ROB, I CAN'T
WAIT FOR THIS PAPER TO COME OUT!
GREAT STORY!
>> ALL RIGHT, WELL, MAKE SURE
I--I WILL SEND IT TO HER AS SOON
AS IT COMES.
[LAUGHTER]
>> OKAY.
>> WELL, THANKS AGAIN ROB.
>> THANKS, EVERYBODY.
>> NICE SLIDE LECTURE AND THANKS
EVERYBODY FOR ATTENDING.
>> YES AND THANK YOU VERY MUCH
FOR THE FINE TALK AND FOR THE
VIEW OF WHAT IT'S LIKE DOWN IN
THE SOUTHERN USA.
>> ALL RIGHT, THANKS SO MUCH AND
YOU ALL BE SAFE AND HAVE A GOOD
DAY