WELCOME TO THE JUNE EDITION OF
THE NIH DNA REPAIR INTEREST
GROUP VIDEO CONFERENCE SERIES.
TODAY I WILL BE CO-HOSTING THE
SEMINAR ALONG WITH KEN KRAMER
AND OUR SPEAKER TODAY IS SHAN
YAN FROM THE UNIVERSITY OF NORTH
CAROLINA AT CHARLOTTE.
BEFORE WE BEGENERATED I WANT TO
ACKNOWLEDGE THE HELP WE GET WITH
OUR SEMINAR SERIES FROM THE
SCIENTIFIC ADVISORY BOARD WHO
HELP US CHOOSE OUR SEMINAR
SPEAKERS AND OUR IT PEOPLE
INCLUDING FELICE HARPER WHO IS
HELPING US WITH AV TODAY.
AND I WANT TO REMIND YOU ALL
THAT MANY PREVIOUS TALKS IN THIS
SERIES CAN BE FOUND ON OUR VIDEO
ARCHIVE.
I ALSO WANT TO REMIND YOU THAT
AFTER TODAY, THE WEBINARS WILL
BE TAKING THE USUAL SUMMER BREAK
NEAR JULY AND AUGUST AND WE WILL
BE ROARING BACK IN SEPTEMBER
WITH A TALK BY JOE MORRIS FROM
THE UNIVERSITY OF BIRMINGHAM ON
THE UK ON MECHANISMS OF
SYNTHETIC LEATHALLITY.
I ALSO WANTED TO TELL YOU THAT
WE ARE SOLICITING NOMINATIONS
FOR STELLAR POST DOCS AND JUNIOR
FACULTY TO PRECEPT SHORT TALK
NOTHING OUR ANNUAL SHOWCASE OF
YOUNG INVESTIGATORS THAT WE WILL
HEAD AS USUAL IN NOVEMBER OF
THIS YEAR.
SO NOMINATIONS SHOULD BE SENT TO
MAY AS SOON AS POSSIBLE WITH THE
NAME OF THE NOMINEE, THE CONTACT
DETAILS AND A SHORT
JUSTIFICATION FOR THE NOMINATION
AND YOU CAN SEND THEM TO ME AT
KARENU@NIH.GOV.
NOW BACK TO TODAY'S SEMINAR,
DR. SHAN YAN GOT HIS Ph.D.
FROM THE UNIVERSITY, AND AFTER
HARVARD JOINED THE FACULTY AT
CHARLOTTE IN 2010.
HE GOT TENURE IN 2016 AND BECAME
FULL PROFESSOR IN 2019.
SHAN DIRECTS THE BIOLOGY
EXCHANGE GROUP THAT WAS ALSO AT
THE BIOLOGY CENTER AND UNC CHAR
LOT, ALSO ASSOCIATE CHAIR FOR
RESEARCH IN THE DEPARTMENT OF
BIOLOGICAL SCIENCES, AND PROGRAM
LEAD IN THE GENOME INTEGRITY AND
CANCER INITIATIVE AT UNC.
HE IS GOING TO TELL US TODAY
ABOUT THE ROLE OF ENDONUCLEACES
IN PRESERVING GENOME INTEGRITY.
BEFORE I HAND THINGS OVER TO
SHAN, I WANT TO REMIND TO YOU
PUT YOUR QUESTIONS TO HIM IN THE
CHAT AND KEN AND I WILL READ
THEM OUT AT THE END OF HIS TALK.
SO I WILL STOPT SHARING MY
SLIDES NOW AND HAND THE FLOOR TO
SHAN.
>> I WANT TO THANK
THE HOSTS AND THE IT PERSON FOR
HOSTING MY SEMINAR TODAY TO HAVE
THIS OPPORTUNITY TO PRESENT WITH
YOU OUR RECENT STUDY IN THE
NUCLEUS 1 AND 2 IN GENOME
INTEGRITY AND CANCER ETIOLOGY.
THIS IS A SHORT OUTLINE, I WILL
GIVE A SHORT INTRODUCTION FOR
THOSE WHO ARE NOT FAMILIAR WITH
APE2 AND 1 IN THE RESPONSE, THEN
I WILL TALK ABOUT APE, AND THEN
I WILL SWITCH TO APE1.
SO [INDISCERNIBLE] CAN BE
GENERATED BY SEVERAL EXOGENIUS
SOURCES, ALL KINDS OF LEADERS
AND IN PARTICULAR WE ARE
INTERESTED IN APE-SIZE AND SSB
TO CALCULATE HOW MANY PER DAY
THERE ARE ROUGHLY
[INDISCERNIBLE] APE SITE AND
55,000 SSB SINGLE STRAND BREAKS
EVEN IN NORMAL MAMMALIAN CELLS
TODAY.
TO REPAIR [INDISCERNIBLE]
DAMAGE, DEPENDING ON THE EXACT
NATURE OF DAMAGE,
[INDISCERNIBLE] PATHWAYS CAN BE
ACTIVELY REPAIR THEM INCLUDING
BASIC EXCISION REPAIR, LET ME
USE MY LASER POINTER HERE,
SINGLE CELL REPAIR PATHWAY TO
REPAIR SSB, AND OTHERS, THE
HOMOLOGOUS COMBINATION AND
COMPARE DSB, AND MMR, AND NER
ARE ALSO COMPARE THEM.
SO NEXT I WANT TO BRIEFLY
INTRODUCE THIS BER PATHWAY TO
YOU FOR THOSE THAT ARE NOT
FAMILIAR WITH THIS.
THE DAMAGE CAN BE RECOGNIZED AND
PROCESSED BY [INDISCERNIBLE] TO
GENERATE APE SIDE, BASICALLY NO
BASICS, THEN THAT CAN BE FURTHER
PROCESSED BY FUNCTIONAL GLUCOSE,
OR APE1 [INDISCERNIBLE] AND THIS
SINGLE STRAND BREAKS SSB CAN BE
RECOGNIZED BY P A RP COMPLEXES
AND XRCC1 AND FOLLOWING THAT
THERE IS I BASIC EXCISION
PATHWAY, THE DIFFERENCE IS
[INDISCERNIBLE] IN WALL,
[INDISCERNIBLE] MEDIATED
PROCESSES.
SINGLE STRAND BREAKS CAN BE
DIFFERENT FROM DNA SUGAR DAMAGE,
BASE DAMAGE AND A
[INDISCERNIBLE] DAMAGE AND THAT
CAN ARREST REPLICATION,
TRANSCRIPTION PROGRAMS OR
EXCESSIVE P A RP ACTIVATION
LEADING TO GENOMICS ABILITY
ESPECIALLY WITH CANCER, AND
NEURODEGENERATIVE DISORDERS.
THE SINGLE STRAND BREAKS CAN BE
LOCALIZED IN NUCLEUS, ALSO
RECENTLY FOUND IN MITOCHONDRIA
AND EVEN IN CYTOPLASMS.
NOW HOW TO REPAIR SINGLE STRAND
BREAKS IS A CHALLENGE
TECHNICALLY AND CONCEPTIONALLY
BECAUSE THE BREAKING CAN CAUSE
RAPID DISASSEMBLY O SO THEY CAN
CAUSE PROBLEM REPLICATION EVEN
WITH DEVELOPMENT OF TECHNICAL
DEVELOPMENT FOR GENOME WIDE
MAPPING OF DNA SINGLE STRAND
BREAKS IN THE GENOME LIKE
[INDISCERNIBLE] CAN MEASURE SOME
TYPE OF SINGLE STRAND BREAKS BUT
NOT ALL TYPES OF DNA SSB, SO
IT'S WORTH IT TO STUDY THE
SINGLE STRAND REPAIR AND
SIGNALING PATHWAYS.
FOR THOSE THAT ARE NOT FAMILIAR
WITH THE DDR PATHWAYS THIS, IS A
GOOD DIAGRAM TO SHOW THE DNA
DAMAGE OR DNA STRESS CAN HANDLE
SIGNALS AND THE SENSED LIKE
MOLECULES LIKE OTHER SENSORS AND
A SIGNAL NODE CAN BE TRANSDUCED
TO DOWN STREAM TRANSDUCERS,
EFFECTORS, EVENTUALLY TO ARREST
CANNED WHAT CAUSE APOPTOSIS
SENESCENSEL, AFTER TRANSCRIPTION
AND REPAIR DNA DAMAGE.
IN RESPONSE TO THOSE,S ARE
ESPECIALLY DOUBLE STRAND BREAKS,
2 KEY PI3 KINASES, ATM AND
[INDISCERNIBLE] TO PREPARE FOR
REPAIR SIGNALING FOR
[INDISCERNIBLE], THE SINGLE
STRAND DNA CAN CODED WITH RPA,
THAT CAN TRIGGER ATR KINASE
ACTIVATION TOGETHER WITH ATR
INTACT PROTEIN COMPLEXES.
THE DDR PATHWAY IS VERY COMPLEX,
BACK IN 1997, WE ONLY KNOW THE
GENE DAMAGE BETWEEN ATRIP, THAT
IS P3 ACTIVATED AND THE ACTIVITY
CAUSE THE CYCLE ARREST, LATER IN
ON IN 2007 IT'S ALREADY SO
COMPLEX, DEPENDING ON THE TYPE
OF [INDISCERNIBLE], THE POSSIBLE
CHOICE TO TRIGGER DDR PATHWAYS.
IN 2017 THIS NICE REVIEW FROM
[INDISCERNIBLE] SUMMARIZED HOW
THIS ATR DDR PATHWAY IS
ACTIVATED.
BRIEFLY IN RESPONSE TO
[INDISCERNIBLE] SINGLE STRAND
BREAK, SINGLE STRAND DNA IS
GENERATED, THE [INDISCERNIBLE]
WILL BE CODED AND THE ATR IS
INTACT [INDISCERNIBLE] COMPLEX
CAN BE RECORDED TO RPA CODED
SINGLE STRAND DNA, THROUGH THE
TECHNICALLY TO AFIB AND RPA.
THEY'RE 2 DIRECT ACTIVATOR
PROTEIN OF ATR CALLED TOP AP1
AND ETWAY 1, BOTH APPROACH WILL
BE RECRUITED TO THAT RP-CODED
SINGLE STRAND DNA SO THAT ATR
KINASE WILL BE ACTIVATED, SO, SO
FAR, TOP 1 AND EETAC1 ARE THE 2
MOST WIDELY RECOGNIZED DIRECT
ACTIVATOR MOLECULES OF ATR
KINASES, WE WILL COME BACK TO
THAT LATER.
SO WHY DO I STUDY ATR/DDR
PATHWAY.
NOW 10 YEARS AGO, MORE THAN 10
YEARS AGO, THERE'S A PAPER SHOWS
THE FIRST ATR INHIBITOR,
[INDISCERNIBLE] CAN SELECTIVELY
KILL CANCER CELLS AND IN
CLINICAL TRIALS THE FIRST
REPORTER CAME IN 3 YEARS AGO,
THE PHASE 1 CLINICAL TRIALS KIND
OF A MORE DEVELOPED BURDEN,
TARGETING KINASES, WITH VERY
ENCOURAGING NEWS ON TARGETING
ATR DDR FOR CANCER THERAPEUTICS.
THERE ARE ALSO OTHERS AT
DIFFERENT STATUS OF CLINICAL
TRIALS.
NOW I WANT TO INTRODUCE SOME
BACKGROUND OF MY 2 FAVORITE
PROTEIN, APE 1 AND APE 2.
SO DR. [INDISCERNIBLE] AND
DR. WILSON POSTED THIS REVIEW ON
APE1, SO THIS IS PRESCRIBING
NEBT APENUCLEUS 1, IT HAS END
TERMINAL DOMAIN,
[INDISCERNIBLE], IT AWLINGS ARE
HAS ASSISTANCE UP TO 5 REMEDIATE
REDOX ACTIVITY AND AS THE
ENDONUCLEUS, PHOSPHOR
DISCIPLINARY FORUS MEDIATED
ACTIVITY, SO IT'S MAINLY
FUNCTION IN THE PAIR, IN
PROCESSING AND TRANSCRIPTION OF
APE1 FROM LITERATURE.
HOWEVER, IF YOU LOOK AT
LITERATURE, TARGETING APE 1 IN
THE TREATMENT OF HUMAN DISEASES,
THERE'S ONLY 1 IN CLINICAL
TRIALS.
APX 3330 TARGETING IS REDOX
FUNCTION, EVEN THOUGH--IT'S WELL
KNOWN FOR FUNCTION IN DEAN
REPAIR, BUT IT'S MECHANISM FOR
OUR METABOLISM SINCE THE PLOT
INDUCED THE [INDISCERNIBLE] AND
GENE DAMAGE RESPONSE IS NOT VERY
CLEAR.
EVEN THOUGH WE HAVE PUBLISHED
LIKE 1500 PAPERS, ON APE1 IN PUB
MED SO THERE'S A LOT OF
[INDISCERNIBLE] MECHANISM FOR
APE1.
NOW I WILL MOVE ON TO APE2.
APEINTERNUCLEUS 2, IN MANY
DIFFERENT SPECS MAY HAVE
DIFFERENT NAME, CALLED MPN2, OR
ONLY LIKE 70-80 PAPERS IN PUB
MED SO FAR.
IT ALSO HAS EEP AT THE END
TERMINUS, AND AT THE SAME
TERMINUS IT HAS OTHER 2 FACTOR
DOMAIN, 1 IS CALLED PAPER
[INDISCERNIBLE] MEDIATED PC
INTEX, FOR THE MOTIF.
ALL THIS 3 FUNCTION MOTIFS ARE
CONSERVED DURING EVOLUTION, FROM
DIFFERENT SPECIES OF APE TO
[INDISCERNIBLE], AND WE
SUMMARIZE THIS APE TO
FUNCTIONALITIES IN GENOME AND
EPIGENOME INTEGRITY MAINLY IN
DNA REPAIR, IMMUNE RESPONSE
EPIGENETIC REGULATION.
OR RECENT STUDY FUNCTION--I
THINK 4 OR 5 STUDIES HILIGHTS
THE APE 1 OR 2.
THIS IS THE FIRST WOIN CAME TBR
STEVE'S LAB AT HARVARD MEDICAL
SCHOOL USING CRSPR SCREENING OF
GENOME WIDE BRCA 2 DEFICIENT
CANCER CELLS.
IT CAME WITH APE 2 AS THE MOST
PROFOUND SYNTHETIC LETHAL FOR
BRCA2 DEFICIENCY AND PROPOSED
APEFUNCTION IN EXCISION REPAIR
MAY BE THE REASON FOR THE
[INDISCERNIBLE].
THEN WILLIAMS PUT TOGETHER A
PAPER NOT ONLY FOR BRCA
DEFICIENT BUT BRCAC1 CELLS, APE2
IS THE EFFECTIVE LETHAL TARGET
AND ALSO PROPOSE APE DYSFUNCTION
IN THE RESOLUTION OF
[INDISCERNIBLE].
THIS YEAR, FROM 2003,
[INDISCERNIBLE]'S LAB PUBLISHED
THIS CELL PAPER REPORTING THAT
THE FUNCTION OF APE2 IN THE MMEG
R J ACTIVITY ESPECIALLY IN THE
RESOLUTION OF THE 3 PRIME OF 3
FLAT STRUCTURES THROUGH IT'S EEP
DOMAIN.
SO ALL THOSE GENOME WIDE STRING
POINTING OUT THAT THE
SIGNIFICANCE OR APE1 OR 2, AS A
SYNTHETIC TARGET BRCA 1 AND 2
DEFICIENT CELLS.
SO IF YOU LOOK AT APE1 AND 2
STRUCTURE, THEY ALL SHARED THE
EEP DOMINANT, THE END TERMINAL
HAS KRAMER FRAGMENT AS
MISINTERPRETED DOMAIN, REDOX
[INDISCERNIBLE] AND EXTRA DOMAIN
AS C-TERMINUS, IF YOU LOOK AT
THE ACTIVITY, APE1 HAS VERY
STRONG OR FAST APEIN THE NUCLEUS
BUT WERE WE GO SLOW
[INDISCERNIBLE] ACTIVITY, APE
WILL KNOCK OUT THE BRCA LETHAL
AND APE2 AND OPPOSITE, IT HAS
WEAK OR SLOW APE IN THE NUCLEIC
ACTIVITY BUT A STRONG OR FIRST
NUCLEIC ACTIVITY.
THE APE2 KNOCK OUT IS NOT LETHAL
BUT HAS SOME DEFICIENCY IN THE
CELL CYCLE.
SO WHEN I SEARCH A WEBSITE, I
SAW THIS APE1, AND OLDER BROTHER
AND YOUNGER BROTHER AND APE1 MAY
SAY OH BROTHER, I WAS DISCOVERED
FIRST SO I CAN DO EVERYTHING
BECAUSE IF YOU CAN SEE, YOU CAN
FIND 1500 PAPERS IN PUB MED, BUT
APE2 MAY SAY OH REALLY, MAYBE I
CAN SEE THAT YOU DON'T EVEN
THOUGH THERE ARE ONLY 70 PAPERS
IN PUB MED.
SO TO FIGURE OUT WHAT APE1 TO DO
TO [INDISCERNIBLE] STABILITY IN
CANCER, WE PRIMARILY USE THIS AS
A MODEL SYSTEM FOR THOSE WHO ARE
NOT FAMILIAR WITH THE SYSTEM,
THIS IS A BRIEF INTRODUCTION SO
XENOPUS, IT'S LOW SPEED
SUPERINATE BUT WITHOUT
RESOURCES.
IF YOU USE HIGHER SPEED, YOU CAN
GET A LAYER OF THE MEMBRANE AND
WE WILL CARVE THIS HSS.
AND YOU ADD A [INDISCERNIBLE]
DNA [INDISCERNIBLE] THAT CAN
FORM A NUCLEAR MEMBRANE, IT CAN
BE REPLICATED, YOU CAN ALSO
ICEOSEALATE THOSE NCLUE I AND
THEN GET NUCLEAR LAYER, CAN YOU
FURTHER SPIN DOWN, YOU CAN GET
MIMIC MP EXTRACTS.
SO LSS, AND HSS, CAN BE USED FOR
STUDY GENE PAIR AND DDR PATH
BASE BECAUSE LSSS, YOU ADD
NUCLEI, AND YOU CAN DAMAGE THE
[INDISCERNIBLE] WITH OTHER GENE
DAMAGING AGENTS CAN ALSO DEPLETE
A TARGETED PROTEIN, OR THE
WILD-TYPE OR MUTANT PROTEIN, CAN
YOU REPAIR OR OTHER THING.
THE GOOD THING FOR HSS, IS YOU
CAN ADD DEFINED STRUCTURES LIKE
PLAZ MIDS MIDDNA WITH THE
DEFINED GENE [INDISCERNIBLE]
THAT YOU CAN LOCATE THE GENE
REPAIR DYNAMICS, DDR PATHWAYS
ACTIVATIONS.
SO LET'S TALK ABOUT THE FUNCTION
OF APE 2 IN ATR [INDISCERNIBLE]
IN CANCER BIOLOGY .D BACK IN
2013 MY GRADUATE STUDENT JEREMY
TO HONORS GRADUATE STUDENTS
[INDISCERNIBLE] TO STUDY WHAT'S
THE FUNCTIONAL APCEREBELLUMS 2
IN HYDROGEN PEROXIDE INDUCE SAYS
ATR PATHWAY USING XENOPUSAS A
SYSTEM.
SO PRIMARILY WE USE H22 TO
DAMAGE CHROMATIN DNA TO CHECK
FOR PHOSPHORYLATION WHICH IS
INDICATOR ISOLATION, WE FOUND
IT'S LOADED TO THE KROEMA TIN
DNA OR TO THE COMPLEXES AND THE
RETINA AS NORMAL COMPLEXES, ALSO
HIGHLY ENRICHED CHROMATIN DNA.
WHEN WE USE DEPLETION, TARGETING
APE 2, IN THE NAP2'S GONE, THE
PHOSPHORYLATION BY IT'S
COMPROMISED, ALL THE ATR,
COMPLEXES, RECRUITMENT IS
COMPROMISED, WHERE WE MAKE AIT
PE RECOMBIN ANT PROTEINS AND WE
FIND THE PHOSPHORYLATION BY H2
IS RESCUED, THE RECRUITMENT OF
APE KROM COMPLEX TO DAMAGED
PROTEINS IS ALSO SKEWED.
SO THAT MEANS APE2 IS CRITICAL
FOR THIS APE 2 FOR THIS REDUCED
ATR DDR PATHWAY IN LSS.
TO FIGURE OUT WHAT'S THE
MECHANISM OF THIS, WE USE 2
TYPES OF MUTATIONS.
ONE IS IN THE PAPER BOX THAT MAY
MEDIATE THE [INDISCERNIBLE] WE
ALSO TESTED TO 2 NUCLEUS
DEFICIENT MUTANT E34 A OR
[INDISCERNIBLE] MUTATION.
SO COMPARED TO THE WILD-TYPE
THIS MUTATION CANNOT RESCUE
CHECK 1 PHOSPHORYLATION BECAUSE
IT CANNOT SUCCESSFULLY RECRUIT
IT FROM THE DNA, THERE'S NO
RECRUITMENT TO ATR COMPLEXES TO
DAMAGE CHROMATIN EITHER, WHEN WE
COMPARE THE NUCLEUS DEFICIENT
MUTATION, WE FOUND WE CANNOT
RESCUE [INDISCERNIBLE], EVEN
THOUGH THE RECRUITMENT TO THE
CHROMATIN DNA HERE IS SAME AS
WILD-TYPE BUT BECAUSE OF THE
DEFICIENCY OF THE NUCLEUS, WE
CANNOT RESCUE THE ATR COMPLEX OF
THE CHROMATIN DNA.
SO THERE ARE 2 MECHANISMS, 1 IS
[INDISCERNIBLE] INTACT FOR THE
BOX, IMPORTANT FOR RECRUITMENT
AND THEN IT'S EXNUCLEIC ACTIVITY
IS IMPORTANT FOR THE DRIVE
DIRECTION.
SO THE MECHANISM FOR THE APE2,
THE ATR, DDR POGHT WAY.
NEXT WITH SCOTT WILLIAMS FROM
NIEHS, VISITED US BACK IN 2000,
2013 OR 14, WE CAME TO
COLLABORATE ON THIS PROJECT AND
WE FOUND THIS VERY UNIQUE MOTIF,
SO MY STUDENT ZACH AND LIN IS
WORKED ON THIS PROJECT BECAUSE
THIS IS ROUGH LOW IN 100 GENES
OR METABOLISM PROTEINS INCLUDING
NIO3, TOP 3 A, SO THEY ARE
HIGHLY CONSERVED IMMUNO ASSAYS.
AND IN THE APE2 IN DIFFERENT
SPECIES, THEY'RE ALSO HIGHLY
CONSERVED.
SO SCOTT IS EXPERT IN STRUCTURE
BIOLOGY SO HIS LAB CRYSTALLIZE
THIS APETO THE FGF MOTIF, IT HAS
UNIQUE HELIOS POSITIVE
EXCITATORY AND ISO HAD 3 BETA
SHADE FORMING THIS WIDE-LIKE
STRUCTURE AND MORE IMPORTANTLY
THIS CAN PREFERENTIALLY BIND TO
SINGLE STRAND DNA AND 1 IMMUNO
ASIGNIFY TO THE MUTATION CAN
COMPROMISE AND PAIR THESE SINGLE
STRAND DNA BINDING.
WHEN WE LOCATED THE X-ACTIVITY
OF THIS USING THIS AS A
SUBSTRATE--OR FILE THE MUTATION,
IT CANNOT BE RESCUED AND COMPLEX
CANNOT BE RESCUED SO THIS
INDICATE THE MOTIF WITHIN THE
AP2 IS CRITICAL FOR IT'S AXO
ACTIVITY AND ALSO IMPORTANT IF
YOU ARE FOR THE ATR DDR
ACTIVATION.
SO THERE ARE 2 QUESTIONS HERE.
WE USE PRIMARILY SO HYDROGEN
PEROXIDE, NOT ONLY IN SINGLE
STRAND BREAKS COULD BE OTHER
TYPES OF THESE LESIONS AS WELL
SO WHAT HAPPENS FOR THE
[INDISCERNIBLE] OF THESE
STRUCTURES?
SO SECOND QUESTION IS HOW IS
THAT SINGLE STRAND GENERATED IF
THIS IS CRITICAL FOR ACTIVATION
OF ATR NUCLEUS ACTIVITY.
TO ADDRESS THE FIRST QUESTION,
BOTH THE POAT DOC IN THE LAB TRY
TO ANSWER THOSE 2 QUESTIONS.
DOES A SINGLE STRAND BREAK
TRIGGER DDR PATH ACTIVATION,
WHAT'S THE MOLECULE OR MECHANISM
OF THIS AS BEING INDUCED FOR THE
DDR PATHWAY.
WE PLASMA BASED USE THIS ENZYME
CAN CREATE SINGLE STRAND BREAK
ON THE TOP STRAND AT THE FINAL
LOCATION WE USE THE PH OS, OF
THE PHOSPHATE SO BOTH ARE OH ON
THE 3 AND 5 SIDE.
SO THIS IS CALLED CONTROL PLAZ
MIDS MID, THIS IS SSB PLAZ MIDS
MID, SO IF YOU USE THE ENZYME TO
LINEARIZE THIS, WE CALL THIS
STRUCTURES BECAUSE THIS ENZYME
GENERATE NYK CANNOT BE
RECOGNIZED BY THE SBF ENZYME
ANIMAL.
SO THIS IS EXPECTED.
USING THE SSB PLAZ MIDS MID,
COMPARE THIS CONTROL PLAZ MIDS
MID, WE ADD DIFFERENT DOSES FOR
THESE S, B COME INTO HS WE SEE.
WE SEE DOSE DEPENDENT ACTIVATION
OF CHK 1 ACTIVATION, WE FURGTDER
VALIDATE USING AT TR INHIBITOR,
822 THAT IS COMPROMISED BUT AND
DEPLETED THOSE TECH POINT
PROTEIN, WE FOUND ASB INDUCED
CHECK POINT PHOSPHORYLATION ALL
COMPROMISED, SO WE ARE PRETTY
SURE THIS IS AND THE SSB
PLASMID, FURTHER MORE WE
CHARACTER AND MODEL THE MOTIF,
WITH APE PIVOT BOX, THIS NEW
MODE, WE CALL MODE 2 THAT
MEDIATED BETWEEN THE APE-TO THE
MOTIF TO THE AND THE PCA AND
THIS MODEL 2 INTAKS IS CRITICAL
BECAUSE C-4708 WILL LOSE THE
INTAKS--REPAIR BACK TO THE
DOUBLE STRANDA PLASMID DNA
TAKEN--THEY AREY SOUV THE
SENSITIVITY, AND THESE ARE THE
SSB PAIR THAT CAN BE QUANTIFIED,
AND WHEN THAT IS TREATED PERFECT
WITH ATR INHIBITED, THOSE REPAIR
PRODUCTS WILL BE COMPROMISED,
SIMILARLY, WHEN WE DEPLETE APE2,
CHECK 1, ATR CHECK 1 CANNOT BE
ACTIVATED SO THE SSB REPAIR IS
COMPROMISED, WE ADD BACK THE
MAKE APE TO PROTEIN, THEN THE
SSB REPAIR MAY BE RESUMED, SO
THIS IS A QUANTIFICATION FACT,
SO, BOTH ATR IS APE2 ARE
ESSENTIAL FOR SSB REPAIR.
NOW HERE ARE THE MECHANISMS OR
THE SSB PAIR MEDIATED BY APE2.
SO THE SSB CAN RECRUIT APE2,
AFTER RECRUITMENT APECAN BE
ACTIVATED AND ONCE APECHOOSE
ACTIVATED IT CAN PERFORM
ACTIVITY THROUGH 5 DIRECTION,
THROUGH SINGLE STRAND DNA, CODED
WITH COMPLEX, IT WILL BE
RECORDED, THEN PROMOTE SSB TO BE
REPAIRED.
SO SUMMARIZE SO FAR WE HAVE
THERE ARE 3 MECHANISM FOR APE2
FUNCTION HERE.
THE FIRST 1 IS MEDIATED BY APE2,
PAPER BOX INTAKS OF THE
[INDISCERNIBLE] WHICH IS
IMPORTANT FOR THE RECRUITMENT OF
APE2 TO THE DAMAGE SIDE, SECOND
MECHANISM IS APE20, FOR THE
MOTIF, CAN INTACT WITH THE
SINGLE STRAND DNA THAT'S
IMPORTANT FOR ACTIVITY IN THE
ACTIVITY.
THIRD MECHANISM IS, THE APE, TO
EGRF MOTIF INTACT WITH THE
[INDISCERNIBLE] POETIC TEEF,
THAT'S ALSO IMPORTANT FOR ATI
VACTIVATION.
SO THERE ARE NEXT SEVERAL
QUESTIONS RESOLVED HERE SO WE
TRY TO DO MORE BY ANALYSIS TO
ATP TO EXPRESSION IN CANCER AND
WE ALSO WANT TO KNOW HOW DOES
APE CONTRIBUTE TO CANCER
ETIOLOGY?
IS APE A DRIVER OR PASSENGER OF
CANCER?
WE STILL HAVE A LOT OF THINGS TO
DO WITH THIS, THE QUESTION IS
CAN APE BE TARGETED FOR CANCER
TREATMENT.
WE WILL TELL YOU SOME PROGRESS
ON THOSE DIRECTIONS.
IN LITERATURE, APE OR EXPRESSION
HAS BEEN [INDISCERNIBLE]
MULTIMELANOMA MN, THIS IS AP TO
MRN EXPRESSION, IT'S OVER
EXPRESSED IN PATIENTS IN THE
MM LEVEL THIS ALSO SHOWED APE
EXPRESSION OR APE 2.
ANOTHER STUDY SHOWS IN LABOR
CANCER PATIENT, THE LEVEL
EXPRESSION, THE TUMOR COMPARED
IT WITH A NORMAL TISSUE SYSTEM
ALSO OVEREXPRESSED.
MORE IMPORTANTLY, APE TO
OVEREXPRESS OR HIGHLY APE 2
EXPRESSION ASSOCIATED WITH THIS
LOWER OVERALL SURVIVAL RATE IN
CANCER PATIENTS.
SO COLLABORATING WITH MY
COLLEAGUE AT UNC SHE MOVED TO
[INDISCERNIBLE] AND AT
UNIVERSITY AND [INDISCERNIBLE]
WORKED ON THIS DRUG AND FOUND
OUT THAT FROM THE TCGA ANALYSIS,
WE FOUND IN 14 CANCER TYPES
ROUGHLY 17% FREQUENCY OF THE
NORMAL ALTERATIONS, ALSO THOSE
OLTERATIONS INCLUDING GAMES OR
AMPLIFICATION, OR THE HOMOLOGY
DIVISION OF APE2 GENE FROM THOSE
CANCER PATIENTS.
ROUGHLY 120 SOMATIC MUTATIONS IN
2 DIFFERENT CANCER TYPES ACROSS
THE WHOLE CODING GENE, SO APE2
IN DIFFERENT REGIONS INCLUDING
THE [INDISCERNIBLE] ZF GF MOTIF.
WHEN WE COMPARED THE MATCHED
TISSUE, TUMOR TISSUE VERSUS THE
NONMALIGNANT TISSUES WE FOUND 6
CANCER TYPES HIDDEN IN THE
LABOR, [INDISCERNIBLE] ALONG
APE2 IS OVEREXPRESSED IN TUMOR
TISSUES.
AND THE EXPRESSION ALSO
CORRELATES WELL WITH OTHER
IMPAIRED DDR PATHWAYS INCLUDING
[INDISCERNIBLE] P A RP AND
OTHERS.
SO WHEN WE USE PANCREATIC CANCER
CELLS WE LOOK AT THE DIFFERENT
ATR REQUIRING ACTIVATION BY
DIFFERENT STRESSORS INCLUDING
HYDROGEN PEROXIDE,
[INDISCERNIBLE],
[INDISCERNIBLE], ETO, AND THE
INHIBITOR FOR THE
[INDISCERNIBLE].
THE CHECK 1 PHOSPHORYLATION BY
THE STRESSFUL CONDITIONS, AND IN
THE RP2 STUDY ARE COMPROMISED
WITH THE APEPROTEIN IS KNOCKED
DOWN BY SiRNA MEDIATED
APCEREBELLUMS 2 KNOCK DOWN.
WE ALSO QUANTIFIED THE GAMMA TO
THE X, WE FOUND APETO KNOCK
DOWN, THE ELEVATED, AND THEY'RE
UNTREATED OR THE STRESSFUL
CONDITIONS AND ALSO
[INDISCERNIBLE] APE TO SENSITIZE
CANCER CELL TO THOSE 4 DIFFERENT
TYPES OF DRUGS OR STRESSFUL
CONDITIONS.
AND TO MOVE THIS FORWARD, WE ARE
INTERESTED IN THE APPLICATION OF
SPECIFIC INPIB HIB THORSITTOR
FROM 1 PAPER PUBLISHED IN 2014,
THERE'S SEVERAL DRUGS,
IDENTIFIED TO INHIBIT ATR DDR
PATHWAY, SO WE MADE HYPOTHESIS,
WHILE IT'S CLOSE TO STRUCTURALLY
THE [INDISCERNIBLE], WE TEST
WHETHER CELASTROL CAN
IBT--INTEGRATE HIB THORSIT THIS
SSB INDUCED SYSTEM, WE ALSO
FOUND THIS CELASTROL CAN INHIBIT
THE MOTIF COMPLEX OVER THE
SINGLE STRAND DNA, ALSO
CELASTROL INHIBITS THE, DOSE
DEPENDENTS THE ACTIVITY.
SO WE IDENTIFY CELASTROL IS THE
FIRST KNOWN COMPOUND INHIBITOR
OF APE, 2, WE ALSO TEST WHETHER
THE CELASTROL TREATMENT CAN
SYNTHESIZE TREATMENT OR CANCER
CELLS TO THOSE STRESSFUL
CONDITIONS, TO INCLUDING
KD--SALLY WHARKS CPT AND THE
ETO.
FURTHER MORE I COLLABORATE WITH
[INDISCERNIBLE] FROM THE
CLEVELAND CLINIC TO LOOK AT APE
TO FUNCTION IN MOUSE AND THE
MITOCHONDRIA STABILITY.
AS YOU KNOW, SIS PLATIN CAN
CAUSE ACUTE INJURY CALLED AKI,
IT'S A SIDE EFFECTS OF THE
TREATMENT, IT WAS IMPORTANT WE
FOUND INDUCED AKI, IF MEDIATED
FROM APE2 UPREGULATION.
SO THIS IS A DIAGRAM TO SHOW THE
PROBLEM CISPLATIN HAPPENS AT A
UNIQUE [INDISCERNIBLE] CALLED
PROXIMAL TUBAL CELL IN
[INDISCERNIBLE], IN THOSE CELLS
SIT CISPLATEIN TREATMENT CAN
CAUSE APE2 UPREGULATION AND CAN
CAUSE CROSS LINK DAMAGE IN DNA
WHERE INTERESTINGLY THE
UPREGULATED APE CAN GET INTO THE
MT AND INTACT WITH KNOWN MUSCLE
MOUSE IN PROTEIN CALLED M1 A9.
THAT LIKELY IS THE REASON FOR
CISPLATIN INDUCED AKI.
THIS IS NORMAL PATIENTS VERSUS
AFTER THE TREATMENT OR THE CIS
PLATIN API, THE IT'S
UNREGULATED, OVER HERE IS
CISPLATIN TREATMENT IN THE MOUSE
THAT TREATMENT CAN CAUSE APE TO
UPREGULATION FOR QUANTIFICATION
BUT NOT APE1 UPREGULATION.
THE [INDISCERNIBLE] GROUP HAVE
THIS KIND OF APE EXPRESSION,
TRANSGENIC MICE SO THIS IS THE
NORMAL CONTROL, THIS IS APE
OVERXRETIONZ, GLOBAL EXPRESSION
THAT CAN CAUSE MORE EARLY DEATHS
OF THE MOUSE, AND WHEN WIEW LOOK
AT THE MOUSE ANALYSIS, COMPARE
WITH THE CONTROL VERSUS APE TO
OVEREXPRESSION, THEY FOUND THE
EXPRESSION CAN CAUSE MORE
CASPACE 3 AND GAMMA
[INDISCERNIBLE] INDICATE MORE
DNA DAMAGE OR CELL DEATH.
WE LOOK AT THE MILD DON DID
YOUIT STRUCTURE WE FOUND THIS
NORMAL MICE, NORMAL MITOCHONDRIA
MORPHOLOGY, FOLLOWING THE APE
OVEREXPRESSED BECOMES ABNORMAL.
CISPLATIN CAUSED [INDISCERNIBLE]
OR OVEREXPRESSION CAUSED MORE
LOCALIZATION OF APETO BE COX4,
OF A MITOCHONDRIA EXPRESSIVE
MARKER AND THE EXPRESSION ALSO
CAN CAUSE MORE ENRICHED
RICHED
COLOCALIZATION.
SO THIS IS A SUMMARY HERE, THIS
IS THE MOTIF THAT ARE REQUIRED
FOR EXPRESS-DUCED ATR, DDR
PATHWAY AND APE 2 TOAXON
NUCLEACE ACTIVITY IS REQUIRED
FOR SSB INDUCED ATR AND DDR
PATHWAY AND THE SSB PAIRS, THE
FUNCTIONAL APE TO ATR, DID, DR
AND I CAN TARGET FOR CANCER
TREATMENT.
SO WE PROPOSE APETO SIGNIFICANT
FOR THAT, FOR--ESPECIALLY IN
BRCA 1 AND 2 DEFICIENT CELLS.
THAT IS A SUMMARY OF OUR
PUBLICATIONS OF APE OVER THE
YEARS.
NEXT I WILL SWITCH GEAR TO APE1,
THE OLDER BROTHER, HOW APE
FUNCTION AS SSB INDUCED ATR, DDR
AND HOW THAT MEDIATE ATR
SIGNALING AND UNEXPECTED
FUNCTIONING IN THE ALL THE DDR
ACTIVATION.
SO I SHARE WITH YOU, APE IS
RECRUITED AND ACTIVATED AT
SSB SITE, FOR THE TUITION, WE
HYPOTH SIZE THAT APE 2 AND APE 1
PLAYED A DIRECT ROLE IN THIS
SIGNALING BECAUSE APE HAS SLOW
OR WEAK ACTIVITY GENERATED THAT
SINGLE STRAND WILL GET.
FIRST OF ALL, THIS STUDY WAS
CONDUCTED BY [INDISCERNIBLE],
AND AN HONORS STUDENT JUDE ON
THIS PROJECT, SO THE FIRST
QUESTION WILL TEST WHETHER WE
DEPLETE APE 1 DOES THAT OOSKT
APE TO RECORD TO THE SITE?
SO WE USE THIS APE TO PREDICT
THE ANTIBODY APE1, SO NO APE
WILL BE REPORTED, TO THE SSB
SITE, THEN APE2 CANNOT BE
RECORDED.
HOWEVER, IF WE DEPLETE APE2,
THEN APE1 STILL CAN BE RECRUITED
TO SSB SITE, INDICATING THAT APE
1 IS UPSTREAM OR APE TO
RECRUITMENT.
SO THIS IS QUITE INTERESTING
BECAUSE P A RP 1 HAS BEEN ISSUED
AS THE SENSOR OF [INDISCERNIBLE]
SO WE USE THIS M-SIZE TO COMPARE
DIFFERENT SUBSTRATES BINDING BY
APE 1.
WE USE DOUBLE STRAND DNA WITH A
SINGLE STRAND BREAK OR A SMALL
[INDISCERNIBLE] SO WE SEE THIS
KIND OF DOSE DEPENDENT ASSEMBLY
OF THE SINGLE STRAND DNA WITH
THIS APE 1 P A RP WHERE THE
SHIFT AFTER BINDING TO SINGLE
STRAIN GAP.
INTERESTINGLY WITH THE GAP
BECOMES 1-3 WITH THE SIDE THEN
THERE'S LESS [INDISCERNIBLE] IF
APE1 AND QUANTIFIED BY MST AS
ALL THIS APE1 BINDING AS A
RESULT.
VERY IMPORTANT TO TRY TO KNOW
WHETHER APE1 CONNECTIVITY IS
ESSENTIAL FOR THIS SIGNALING
PATHWAY.
SO WE IDENTIFY CRITICAL RESIDUE
MUTATED CALLED D306 A WHICH IS
HIGHLY CONSERVED IN THE XEN
OPUS MOUSE, SO THE WILD-TYPE
MAKE APE1 BUT NOT THIS
EXODEFICIENT AND THE COMPLEX
RECRUITMENT TO S'S BESIDES.
AND FOR THE S'S TO BE REPAIRED,
IF YOU DEPLETE IT HAS TO BE
REPAIRED, COMPROMISED BUT WHEN
YOU ADD BACK THE WILD-TYPE AND
NOT THE D MUTANT CAN RESCUE THE
SSB PAIR.
SO APE1 IS FOR THE FOR THE
SIGNALING AS WELL.
INITIATED THE INTERSECTION FOR
THIS, TO GENERATE THE STEP 1.
INITIATION OR INITIATION.
THE SECOND STEP IS APE2 IS
RECRUITED AND ACTIVATED BY THOSE
3 EARLIER TO CONTINUE THE
INTERSECTION SO YOU CAN GET A
LONGER SINGLE STRAND DNA THAN
ATR COMPLEXES WILL BE RECRUITED
THAT YOU EVENTUALLY FILL THE GAP
OF THE SSB REPAIRS COMLICATE.
SO THIS MECHANISM IS ALSO
RECOGNIZED BY COLLEAGUE, KEITH
IN HIS REVIEW, IN THE CELL
BIOLOGY, TIED TO OTHER SINGLE
STRAND BREAK PAIR FOR THE
[INDISCERNIBLE] DISEASES
PUBLISHED LAST YEAR IN 2022.
OKAY, I WANT TO SHARE ANOTHER
UPDATE, VERY, VERY RECENT UPDATE
OF ATR DDR DR SIGNALING PATHWA.
AS YOU ALL LEARNED AROUND 20
YEARS AGO, THIS IS A LEGENDARY
COMPLEX RECRUITED TO DAMAGE SITE
AND MEDIATED ATR ACTIVATION
BREFLY, EITHER OF THE DSB ENDS
OR [INDISCERNIBLE] SO SINGLE
STRAND DNA WILL BE RECRUITED AND
REPORTED AFTER THE SINGLE STRAND
DNA BECAUSE THE RPA-ATRIP
INTERACTION CAN BE RECRUITED TO
THOSE RPA ENIN THE SITES OR IT
IS [INDISCERNIBLE] SO THIS IS
THE TRADITIONAL LEGENDARY MODEL
PROPOSED BY ZOU AND ELLEDGE 20
YEARS AGO.
BUT INTERESTINGLY FOLLOW UP
STUDY FROM OTHER LABS, INCLUDING
[INDISCERNIBLE] AND OTHERS SHOW,
NOT ONLY RPE DEPENDENT BUT ALSO
THE ATR TRIP RECRUITMENT DAMAGE
SITE COULD ALSO BE RPE
INDEPENDENT.
SO WHAT'S THE PROTEIN THAT CAN
BRING ATR COMPLEX TO ASSESS DNA
IN THE RPA INDEPENDENT MANOR FOR
ACTIVATION, HAS BEEN AN
OUTSTANDING QUESTION FILLED FOR
ALMOST 20 YEARS.
SO WE PRODUCE OR WE HYPOTHESIZED
INITIALLY THAT AP1 CAN
[INDISCERNIBLE], THIS BASED ON
OUR COMPLETION EXPERIMENT SO WE
USE DOUBLE STRAND DNA WITH
SINGLE STRING GAP, SO WE
DESIGNED THIS WITH BIOTIN ON THE
TOP STRAIN, THESE 13 NUCLEAR
TIDE GAPS SO WE ADDED THIS THE
STRUCTURE WE CALL IT SINGLE
STRAND DNA GAP STRUCTURES INTO
HHS, INTERESTINGLY, BOTH THE 13
NUCLEAR SINGLE STRAINED GAP AND
THE OTHER GAP FOR 1
PHOSPHORYLATION, ALSO WE SEE
RECRUITMENT RP COMPLEX, RP 70,
RECRUITMENT FOR THE STRUCTURES,
WE ALSO SEE APE1 RECRUITMENT AS
WELL SO NOT SURPRISINGLY IT WAS
ALL RECOTTERRED, RIGHT.
MORE IMPORTANT WHEN WE REDEPLETE
APE1, AND PHOSPHORYLATION, IT'S
COMPLETELY GONE, AND THAT'S IN
EFFECT RECRUITMENT RPE70 OR NOT
EVEN OLD RPE32, BUT THE
DIFFICULT FIND SUGGEST ATRIP
CANNOT BE RECORDED WITHOUT APE1.
SO THIS IS QUIED INTERESTING
SUGGESTING THAT APE1 COULD BE
THE GUY THAT WE'RE LOOKING FOR
AROUND 20 YEARS.
SO 1 OF THOSE WAS CAN YOU
REVISIT RPA DEPLETION, OKAY, SO
WE DID RPA DEPLETION AND WE
FOUND THAT IT DEPLETED RP A 32,
AND CHECK FOR POSFORALATION BUT
OUR RPA ANTIBODY CUT SOME OF THE
PROTEIN HAD HAS, SO THIS IS COB
CYSTENT WITH PRIOR PUBLICATION
BY KD--SALLY LAWEDDER DALE LAB.
BUT IMPORTANTLY, WITH THAT RPA,
[INDISCERNIBLE] 32, APE 1
BINDING 2 SSDNA GAPS ARE STILL
THERE, AND SOME ARE THE PROTEINS
CAN STILL BE RECRUITED TO THE
SSB [INDISCERNIBLE] STRUCTURES.
SO WE QUANTIFY THE ATRIP ON
Bs, VERSUS WE [INDISCERNIBLE]
ATRIP, WE FOUND YES IT IS
REDUCED BUT VERY MINOR, LIKE
20-30% REDUCTION.
SO THIS COULD BE RPA INDEPENDENT
BUT AE1 DEPENDENT RECRUITMENT OF
OUR ATRIP COMPLEX TO SINGLE
STRAND DNA.
FURTHER MORE, IT'S VERY
INTERESTING WE FIND THAT APE1,
SO THIS IS TAG APE 1, THIS IS
GST, COMBINED TO THE
[INDISCERNIBLE] ATE GUESTS WE
HAVE [INDISCERNIBLE] IN THE
INVITRO SYSTEM, AND THIS APE
INTACT WITH A SINGLE STRAND DNA
IS A LENGTH DEPENDENT, ALSO
CHARACTERIZE THE DOMAIN
REQUIREMENTS WITH THIS, SO IF
YOU COMPLETED THE FIRST 34, THE
MISTRESS [INDISCERNIBLE] ATPE1
YOU LOSE THE BINDING WITH SINGLE
STRAND DNA.
HOWEVER THAT END TERMINAL IS NOT
ENOUGH FOR THIS BINDING.
WE'RE INTERESTED IN APE ALSO
INTACT WITH THE [INDISCERNIBLE]
PROTEIN AND I WANT TO POINT OUT
THAT THE END TERMINAL PROTEIN
DELETION CANNOT INTACT WITH THE
SINGLE STRAND DNA AND 100 IMMUNE
ASSAY DIVISION CANNOT INTACTIVE
WITH THE ATRIP SO WE
CHARACTERIZE THIS 2 TRAG
FRAGMENT APE1.
SO WE WANT TO KNOW WHETHER
APE IN ITSELF CAN RECRUIT TOW
SINGLE STRAIN DNA GAP
STRUCTURES.
YES WHEN WE ADD THE SNA, AND THE
IT CAN BE RECRUITED AND WE
DEPLETED THE FIRST 100 IMMUNO
ASSAYS THAT CANNOT INTACT THE
ATRIP BUT STILL BIND TO SINGLE
STRAND DNA AND CANNOT
[INDISCERNIBLE] ATRIP.
IF YOU COMPLETE THE FORM, CANNOT
INTACT WITH THE SINGLE STRAND
DNA EVEN THOUGH INTACT WITH
[INDISCERNIBLE] ATRIP COOPERATE
BE ABLE TO RECRUIT HIS ATRIP TO
SINGLE STRAND DNA SO THIS IS
STILL THINKING WE EXTRACT, WE
DEPLETE APE1, BUT AT WILD-TYPE
THE MUTANT, THIS LIMITATION
CANNOT RESCUE ATRIP BINDING THIS
MUTATION CANNOTRESS CUE ATRIP
BINDING EITHER.
WE ALSO TESTED THIS IS INDEED
NUCLEUS INDEPENDENT BECAUSE THE
MUTATION [INDISCERNIBLE] IS INDO
MUTANT DEFICIENT HAS NOTHING TO
DID WITH THE RECRUITMENT TO
SINGLE STRAND DNA.
MORE INTERESTING AND FURTHER
MOIRE WE LOOK AT THE APE1
POTENTIAL INTAKS WITH RPA, WE
FOUND THE 2 POTENTIAL MOTIF IN
THE APE1.
ONE IS THE D69, ANOTHER IS THE
E48/49 MUTATION CALLED ARM
BINDING MOTIF 1, ARM BINDING
MOTIF 2.
SO A MUTANT EACH OF THEM CAN
CAUSE SOME SORT OF INTERACTION
RPEINTERACTION REDUCTION BUT A
MUTANT WILL DECREASE
BOTH--INCREASE BOTH
SIGNIFICANTLY INTACT WITH RPA.
COME BACK TO THIS RPA MEDIATED
[INDISCERNIBLE] DOUBLE STRAND
DNA--YES, IT'S SUFFICIENT TO
RECRUIT ATRIP IMPORTANT TO
SINGLE STRAND DNA AND THIS GST
ALSO CAN DO THE SAME THING BUT
IF YOU HAVE BOTH, THE
COMBINATION INDICATE IT'S NOT
DEPENDENT ON EACH OTHER, IT'S
NOT SINNER GESTIC SO WE PROPOSE
THE MODEL IN THE COMTENT OR
RECONSTITUTION SYSTEM, BOTH RP
AND ATRIB CAN DO THE RECRUITMENT
OF ATRIP INDEPENDENTLY, AND
THERE ARE APUINDEPENDENT IS
RPEINDEPENDENT AND
RPEINDEPENDENT.
SO THAT MEANS ATRIP CAN RECRUIT.
IT CAN RECRUIT ATRIP TO THE
SINGLE STRAND DNA.
HOWEVER, IN THE RP DEPENDENT.
SOMEHOW AP-1 MAY HAVE TO RELEASE
INHIBITORY PROTEIN X THAT
REGULATES RECRUITMENT THROUGH
THE RPA DEPENDENT MECHANISM.
SO I WILL GIVE YOU A LITTLE BIT
OF THE LAST STORY WE RECENTLY
PUBLISHED ON THE APS FUNCTION IN
THE [INDISCERNIBLE] AS YOU KNOW
IN THE NUCLEI, IT'S VERY UNIQUE
COMPARTMENT WHERE THE RBA IS
TRANSCRIBED INTO RIBOSOME RNA.
THIS IS A VERY UNIQUE REGION TO
FORM [INDISCERNIBLE] THROUGH
DIFFERENT MULTIPLE
[INDISCERNIBLE] MECHANISM CALLED
LIQUID OF LIQUID SEPARATION.
WE HAVE MARKERS, I WANT TO POINT
OUT THIS GC COMPONENTS, MPM 1
INDICATE THE DC COMPONENT OF THE
NUCLEUS.
THIS NUCLEI CAN BE STRESSED BY
DIFFERENT TYPES OF DAMAGE
INCLUDING DOUBLE STRAND BREAKS,
INHIBITION, STRESS, OSMATIC
STRESS, TRIGGER ATN AND ATR DDR
PATHWAYS AND MORE REEBTLY THE
APS MEDIATED BY THIS TOPIC P1
AND OUR STUDY ON APE1 CAN
ILLUSTRATE THE DIFFERENT
MECHANISM AND HOW ALL OUR
PATHWAYS ARE ACTIVATED.
SO, WHY WE STARTED APE 1 IN APS,
BECAUSE AP1 HAS THIS MISTRESS
INTERNAL DOMAIN CALLED THIS IDR
IN PLACE OF THIS OLDER MOTIF.
ALSO MEDIATED ARE INTACT BUT
WHEN WE DID OUR STUDIES IN DEAD
WHAT WE FIRST TRIED TO SAY
WHETHER KNOCK DOWN APE1 CAN
AFFECT H2 TO INDUCE
PHOSPHORYLATION, THAT'S THE
CASE.
WE ALSO USE APETHE BEST
INHIBITOR MODE CALLED APE 3,
DOZE DEPENDENT AR3 CAN CAUSE
REDUCTION OF CHECK 1
PHOSPHORYLATION, BUT
[INDISCERNIBLE], THEN MY POST
DOC DO THE OVEREXPRESSION OF
YP/AP1 THING AND HE FOUND YFP A
P1 CAN CAUSE PHOSPHORYLATION
UPREGULATION BUT SURPRISINGLY HE
FOUND ALL THE MUTATIONS HE
TRIED, THE AXO DEFICIENT OR THE
EDO EXO, OR ALL CAN CAUSE CHECK
1 PHOSPHORYLATION, AND
NONCONDITIONS.
SURPRISE, SO THEN WE TEST
WHETHER IN THE P A MP1 WHETHER -
TRACKS WITH THE NUCLEUS, IF WE
ADDED THIS RECOMBIN ANT PROTEIN,
WE CAN FORM THIS BI MOLECULE, IF
WE REMOVE END TERMINAL DOMAIN,
CANNOT DO IT, WELL, WE ADDED
THOSE PROTEIN INTO THE SPS
BUFFER, IT CAN GROW AND FUSE
INTO A LARGER [INDISCERNIBLE],
AND THE NUCLEIC EXTRACT WE FOUND
THIS END TERMINAL DELETION FEELS
TOAC IV THERAPY POSFORALATION,
THE PULL DON ASSAY, SHOWS THIS
END TERMINAL IS VERY IMPORTANT
FOR INTAKS WITH ATR COMPLEXES
WITH THE NUCLEIC CONSTRUCTS.
WE LOOK AT IMAGING OF THE YFAPP,
WITH THE WILD-TYPE APE1 CAN CO
LOCALIZE IN THE NUCLEUS AND THE
BOTH ATR APE1 CO LOCALIZED WITH
THE YFP 11.
THOSE IN THE CANCER CELL, BUT WE
COULDN'T FIND THOSE IN NORMAL
CELL.
AS YOU KNOW, THERE ARE 2
ACTIVATOR TOPICS AND ETA, AND 1
FOR KINASES, WE FOUND HAVING
INTERESTING DOMAIN IN APE1 AS IT
CONSERVE DOMAIN AND THE
TRANSCRIPT O FAN 9119
R-MUTATION, NOW ALSO IN THE END
TERMINAL WHILE I COMPARE THOSE
RECRUITMENT INTO THE NUCLEUS, WE
FOUND THIS [INDISCERNIBLE] 7
MUTATION CANNOT RECRUIT, OR
CANNOT BE LOCALIZED INTO THE
NUCLEUS.
AND THIS [INDISCERNIBLE], THE 7
R PLURIBUITATION CANNOT TRIGGER
THE PHOSPHORYLATION, THE TRIP TO
1 CAN NOW TRIGGER THAT EITHER.
WE LOOK AT THE BINDING WITH ATR
AND THE FONT, AND THE R-MUTATION
VAIM AS WILD-TYPE CAN BIND TO IT
BUT IT CANNOT FORM THE
[INDISCERNIBLE] IN THE NUCLEUS
EXTRACTS.
MORE IMPORTANT IS HOW WE SEE THE
KINASES IN THIS, WE USE THE IT
AS A SUBSTRATE, WE FOUND THIS
WILD-TYPE APE 1 CHECK 1 BY THIS
MUTATION KOONLT CHECK 1 BY
[INDISCERNIBLE] THIS SO INDICATE
ATR IS VERY ACTIVE IN THE
KINASES.
THIS CAN CAUSE THE SYNTHESIS IN
REDUCTION BUT IT CAN BE REVERSED
BY ATR INHIBITOR, THE CELL
ABILITY ALSO CAN BE REDUCED AND
IT CAN BE REVERSED, THIS IS P53
POPULATION, SO PUTTING THINGS
TOGETHER HERE IS THE WORKING
MODEL THIS IS NOD SURPRISING
BECAUSE THE APE1 HAS
[INDISCERNIBLE] ACTIVITY THAT
ARE IMPORTANT FOR THE HR
SIGNALING AND THE STRESSFUL
CONDITIONS SO WE PRESENT OVER
HERE AND IN CANCER CELLS,
SOMEHOW THIS CAN FORM THE
LIQUID, LIQUID SEPARATION,
THROUGH THEEND TERMINAL, AND THE
TRANSCRIPT O FAN, AND CAN
RECRUIT ATR TRIP, AND INTO THE
NUCLEI, AND ACTIVITY WITH THE
NUCLEI PATHWAY ACTIVATION EMPLOY
SO THIS IS A QUICK SUMMARY, THIS
IS REQUIRES IT TO BE REPAIRED
FOR SIGNALING, APE1 IS INTACT
WITH AND RECRUITED TO SINGLE
STRAND DNA THROUGH
NON[INDISCERNIBLE] FUNCTION,
ALSO PRESENT FOR SHOWING IT CAN
ASSEMBLE BY MITOCHONDRIA, TO
PROMOTE THE DDR PATHWAY IN
CANCER BUT NORMAL CELL.
SO OVERALL APE1 IS ACTIVATOR OF
THE ATR, DID, DR PATHWAYS.
SO PUT THEM TOGETHER, BOTH THE
OLDER AND YOUNGER BROTHER APE1
AND APE2, IN ADDITION TO THE DNA
REPAIR FUNCTION, BOTH OF THEM
ARE ATR, DID, DR IMPORTANT
CONTROL IN ATR DDR PATHWAY.
SO YOU CAN AM BACK TO THIS
SCREEN BY--IN ADDITION TO THE
BER AND THE MMEJ ACTIVITIES THEY
FOUND OUT THAT IT HAS SYNTHETIC
LETHALITY FOR THE BRCA 2
DEFICIENCIES SO LYLELY ATR, DDR
DEPOSIT COULD BE 1 OF THE
MECHANISMS FOR BRCA DEFICIENCY.
SO LASTLY I WANT TO THANK MY LAB
MEMBERS AND COLLABORATORS OVER
THE YEARS AND MY FUNDING AGENCY.
SO THANK YOU FOR YOUR ATTENTION.
WE WILL START HERE.
>> THANK YOU SO MUCH.
THAT WAS REALLY INTERESTING.
WE HAVE A NUMBER OF QUESTIONS IN
THE CHAT, SHOULD I KICK IT OFF,
KEN?
>> GO FOR IT.
>> OKAY, FROM SARAH HINGLE, THIS
IS IN REFERENCE TO YOUR EARLY
SLIDES ON APE 2.
ARE THERE MULTIPLE APE 2S ON
SINGLE STRANDED DID, NA IN YOUR
EMPHASIS, THERE ARE MULTIPLE
BANDS SO I'M CURIOUS WHAT THE
STOICHIOMETRY IS ON THE
SUBSTRATE.
>> SO THE MULTIPLE, CAN REFER
THE QUESTION AGAIN?
>> SHE ASKED IF THERE WERE
MULTIPLE APCEREBELLUMS 2 MEL
COOLS IN THE STRINGLE STRAND DNA
EMPHASIS, SINCE THERE WERE
MULTIPLE BANDS PRODUCED SHE WAS
WONDERING WHAT THE STOICHIOMETRY
WAS?
>> THAT'S A GOOD QUESTION BUT WE
HAVEN'T FIGURED OUT HOW MANY
MOLECULES CAN FORM KIND OF A 1
OR 2 OR MULTIPLE APE TO BINDING
TO SINGLE STRAND DNA YET.
IT COULD BE, THAT'S A
POSSIBILITY BUT WE HAVEN'T
FIGURED IT OUT YET.
>> OKAY, SO SARAH ALSO ASKED OUR
APE2 ASKS IF THEY'RE SENSITIVE
TO KNOCK OUT INHIBITORS.
>> COULD GOOD QUESTION, 1 OF
MANY QUESTIONS.
WE ARE WORKING ON THAT AND I
THINK KD--SALLY -
-[INDISCERNIBLE] IN THE LAB
IS WORKING ON THAT.
GOOD QUESTION.
>> THERE'S A QUESTION FROM
[INDISCERNIBLE],
XENOPUSTPRODUCES DOUBLE STRAND
BREAKS THAT ACTIVATE APM, DO YOU
THINK WHETHER IT'S ISHT
INTERACTS WITH APE2 AND STOPS
STRAND BREAK REPAIR?
>> YES, GOOD QUESTION AS WELL.
WE HAVEN'T TEST THIS QUESTION
DIRECTLY BUT AS YOU KNOW, APE2
PLACE RULE IN MMEJ PATHWAY, WAIT
FOR APE TO RECOGNIZE WHERE THE
DSB ENDS AND PROCESS IT FOR
REPAIR OR SIGNALING IS UNKNOWN,
IT'S UNKNOWN.
HOPEFULLY I CAN TALK TO YOU
ABOUT IN THIS NEXT COUPLE YEARS
>> OKAY, DR. SAKAMORO,
INTERESTING TALK, THANK YOU WHAT
CANCER ASSOCIATED PROTEIN HELPS
APE1 RECRUITMENT TO NUCLEI?
>> THAT'S ANOTHER MILLION
QUESTION, WE ARE VERY INTERESTED
IN THIS FINDING.
WHY IN CANCER CELL WE SAY THIS
APEOVEREXPRESS CAN BE RECORDED
TO NUCLEI, BUT NOT NORMAL CELL.
NOW, WE ARE TESTING APE TO
RECRUITMENT TO NUCLEI USING
DIFFERENT CANCER CELL LINES AND
DIFFERENT NORMAL CELL LINES AND
TRY TO FIGURE OUT WHAT'S THE
EXACT UNDERLYING MECHANISM.
THERE ARE SEVERAL PROPOSED
NECKANISM.
FOR EXAMPLE, IS THERE ANY CANCER
CELL ONCA GENE IN CLUE E I THAT
CAN RECOGNIZE APE, 1 CAN RECRUIT
IT OR ANY UNIQUE STRUCTURE IN
CANCER CELL CAN PROMOTE APE1 TO
NUCLEI.
HOPEFULLY NIH WILL GIVE US MORE
MONEY AND WE CAN FIGURE OUT THE
DETAILS.
[LAUGHTER]
>> ALL RIGHT.
WE SEE THAT APE 2 BECOMES--THIS
IS FROM CAROL SHRADER, WE SEE
THAT APE2 BECOMES
[INDISCERNIBLE] IN GERMAL LYMPHB
LYMPHOCYTES WHERE LYMPH1 IS
GREATLY REDUCED THIS ALTERATION
OF THE RATIO OF APE1 TO 2 MIGHT
BE SIMILAR TO YOUR APE2
TRANSGENIC MOUSE, ALSO APE1 AND
2 WORK TOGETHER IN B-CELLS
DURING ANTIBODY'S CLASS SWITCH
RECOMBINATION, IS ANYTHING KNOWN
ABOUT THE INTERACTION BETWEEN
THE 2 BROTHERS?
>> THAT'S EXCELLENT QUESTION, I
WOULD SAY, IMMUNE CELLS.
THERE ARE LOTS OF STUDIES BY
THEIR GROUP AND I THINK THE
RATIO--FIRST OF ALL I WANT TO
SAY THEY MAY DO DIFFERENT.
THEY HAVE DIFFERENT NUCLEUS
ACTIVITIES, THE EXPRESSION COULD
BE DIFFERENT, OUR LAB HAVE SOME
DATA SHOW ACTUAL INTACT WITH
EACH OTHER.
HOW THEY COMPETED WITH EACH
OTHER, WHO WILL DO MORE, WHO
WILL DO LESS, DEFINITE DEPENDING
ON THE CONTACT EITHER IN
DIFFERENT CELL TYPE, DIFFERENT
TISSUES AND THERE ARE DIFFERENT
STRESSFUL CONDITIONS.
SO MANY POSSIBILITIES.
THERE'S LESS THAN 100 PAPERS ON
APE2.
I HOPE THE NEXT ERA OF APE2 WILL
BE COME NOTHING THE NEXT 10
YEARS.
>> KEN?
>> THAT'S IT.
>> OKAY, THERE'S 1 MORE QUESTION
ACTUALLY THAT GOT ENDED UP BEING
POSTED TO ME PRIVATELY BUT I
THINK IT'SA MEANT FOR EVERYBODY,
IT'S FROM [INDISCERNIBLE] AGAIN
SAYING GREAT TALKS AND DATA
THANK, IF APE2 IS UPREGULATED
AND EXAMINED, WHY?
DOES IT CORRELATE WITH THE
DEGREE OF PROLIFERATION?
AND SECOND QUESTION IS DID THE
APE2 DRUG REGULATION THE
SENSITIVE IV THERAPY TUMORS?
>> WE WOULD LIKE TO BE ABLE TO
ANSWER THOSE QUESTIONS.
WE ARE ACTIVELY PURSUE THESE
QUESTIONS.
I WOULD SAY VERY LIKELY, BUT I
HAVEN'T PULL THOSE DATA
TOGETHER.
BUT, I WANT TO POINT OUT HERE,
THE APE2 BIOLOGY AND IN CANCER
IS VERY COMPLEX.
OUR RECENT REVIEW ARTICLE IN A.
E. R. CANCER FOUND THAT APE2
OVEREXPRESS COULD BE POSITIVE OR
NETIONATIVELY ASSOCIATED WITH
SURVIVAL DEPENDING ON THE CANCER
TYPE.
SO, IN OTHER WORDS, APECOULD BE
[INDISCERNIBLE] COULD ALSO BE A
TUMOR SUPPRESSOR GENE DEPENDING
ON THE CONTACT.
SO WE TRY TO FIGURE OUT
MOLECULAR DETAILS AND REPORT TO
YOU HOPEFULLY SOON BUT DEPENDING
ON HOW NIH DECIDE TO FUND US BUT
I REALLY THANK YOU FOR THE
CRITICAL QUESTION, FIRST OF ALL
FOR SURE.
>> AND THERE'S NO MORE QUESTIONS
IN THE CHAT BUT THERE IS A
COMMENT AGAIN FROM CAROL SHRADER
SAYING I'M GLAD PEOPLE ARE
FINALLY INTERESTED IN APE2,
EXCLAMATION, EXCLAMATION,
EXCLAMATION.
>> THANKS FOR THE COMMENT AND
ENCOURAGEMENT.
>> ALL RIGHT, IF THERE ARE NO
OTHER QUESTIONS, I'M NOT SEEING
ANY, IT JUST LEAVES ME TO THANK
TODAY'S SPEAKER SHAN YAN, THANK
YOU VERY MUCH FOR A VERY
INTERESTING TALK.
OTHER PEOPLE ARE SAYING THANKS
IN THE CHAT, TOO, SO I ALSO WANT
TO REMIND EVERYBODY THAT SHAN'S
TALK WILL SOON BE POSTED ON OUR
ARCHIVE, I THINK USUALLY WITHIN
24 HOURS, OUR IT PEOPLE ARE VERY
GOOD AND ALSO JUST TO WISH YOU
ALL A HAPPY SUMMER AND SEE YOU
HERE IN SEPTEMBER FOR JIM
MORRIS.
>> THANK YOU ALL FOR YOUR
ATTENTION.
>> THANK YOU.
>> BYE-BYE.