>>WELCOME TO THE DNA REPAIR
INTEREST GROUP.
I'M KEN KRAEMER.
MY CO-HOST IS OFF WITH HIS
FAMILY.
YOU KNOW ABOUT THIS BECAUSE YOU
SUBSCRIBE TO THE DNA REPAIR
INTEREST GROUP IF YOU DON'T YOU
SEND EMAIL KRAEMERK@MAIL.NIH.GOV
AND ASK TO BE ON THE LIST IN
JANUARY WE'RE GOING TO HAVE A
TALK ON DOUBLE MECHANISM OF
DOUBLE STRAND BREAKS AND IN
FEBRUARY WE'LL SPEAK ABOUT THE
DNA REPLICATION FOR DYNAMICS
WITH CHEMOTHERAPY RESPONSE AND
IN MARCH WE'LL HAVE MORE ON DNA
RESPONSE ON ALL TELEMERES AND IN
APRIL WE'LL HAVE A WRAP-UP OF
DNA REPAIR MITOCHONDRIA IN THE
NEURODEGENERATIN
NEURODEGENERATION.
WE'LL HAVE A TALK TODAY IS GOING
TO GO FOR THE USUAL TIME BUT IF
YOU FOLKS HAVE ANY QUESTIONS,
PLEASE PUT IT INTO THE CHAT AND
I WILL READ THE QUESTIONS AT THE
END.
AND TODAY'S SPEAKER, DR. KRISTIN
ECKERT IS GOING TO SPEAK ABOUT
DNA POLYMERASE NAVIGATION
THROUGH DIFFICULT-TO-REPLACE
SEQUENCE TO PREVENT GENOME
INSTABILITY AND ASKED HER TO
GIVE A SPECIAL INTRODUCTION
ABOUT HER BACKGROUND AND WHAT
IT'S LIKE TO BE ONE OF THE
PIONEERING WOMEN IN SCIENCE THE
PAST FEW YEARS.
SO WHY DON'T WE TAKE IT AWAY,
KRISTIN.
>> THANK YOU, KEN FOR THAT
INTRODUCTION AND THANK YOU TO
EVERYONE FOR TAKING TIME AWAY
FROM YOUR VERY FULL SCHEDULES TO
JOIN MY PRESENTATION.
TODAY I AM GOING TO TALK ABOUT A
TOPIC NEAR AND DEAR TO MY HEART
AND THAT IS THE TYPES OF GENETIC
VARIATION THAT ARISE IN TUMORS.
MY LAB VERY INTERESTED IN
UNDERSTANDING THE MECHANISMS BY
WHICH VARIOUS TYPES OF GENETIC
VARIANTS ARE CREATED DURING THE
IT PROCESS OF TUMORIGENESIS AND
WE KNOW THEY'RE KEY FOR FUELING
CANCER CELL EVOLUTION.
THE TYPES OF GENETIC VARIATION
IN OUR STUDIES RANGES FROM
REPETITIVE SEQUENCES WITHIN THE
GENOME TO MECHANISMS OF LARGE
STRUCTURAL VARIATION.
AND AS KEN MENTIONED, I PUT
TOGETHER THE BEGINNING OF MY
PRESENTATION IN A DIFFERENT WAY
TO INTRODUCE YOU TO NOT ONLY
THIS TOPIC BUT TO MYSELF.
SO I ACTUALLY AM FROM A SMALL
TOWN IN NORTHERN PENNSYLVANIA
AND LIKE MANY STUDENTS I LOVED
MY BIOLOGY AND CHEMISTRY CLASSES
BUT WHEN IT WAS TIME TO GO TO
SCHOOL IT WAS ONLY ONE CHOICE
PENN STATE WHICH WAS VERY LITTLE
TUITION AND THE STATE SCHOOL.
DURING MY COURSES TO BE BECOME A
MEDICAL TECHNOLOGIST WHICH WAS
MY ORIGINAL TRACK THIS IS SCHWAB
AUDITORIUM AND A GENETICS CLASS
OF ABOUT 600 STUDENTS AND WHEN
IT CAME TO THE TOPIC OF FRUIT
FLIES AND MUTATIONS I FELL IN
LOVE WITH THE CONCEPT THE GENOME
COULD CHANGE AND GENETIC
MUTATIONS HAPPEN AND THIS IS AN
ACTIVE PROCESS.
I HAD A VERY ASTUTE FACULTY
MEMBER WHO INTERVIEWED ME AND
TOLD ME YOU DON'T WANT TO BE A
MEDICAL TECHNOLOGIST, YOU WANT
GO TO RESEARCH TO WHICH I
ANSWERED WHAT IS RESEARCH.
AT THAT TIME I HAD NO CLUE.
I DIDN'T KNOW WHAT GRADUATE
SCHOOL WAS MUCH LESS GETTING A
Ph.D..
SO HE DIRECTED ME TO A LAB WHERE
I STUDIED BACTERIA DURING MY
SENIOR YEAR IN COLLEGE AND
ISOLATED MUTANT FORMS THAT COULD
NO LONGER FIX NUTRIGEN AND I
JOINED THE LAB FOR CANCER
RESEARCH AT McCARDLE LAB AND
HERE I WAS ABLE TO STUDY
MUTATIONS IN THE CONTEXT OF A
PERTINENT DISEASE OF CANCER.
AT THE TIME I JOINED GRADUATE
SCHOOL IN 1982 THE WAR ON CANCER
WAS RELATIVELY YOUNG WE KNEW
THERE WERE SPECIFIC THINGS IN
OUR ENVIRONMENT THAT CAUSED
CANCER AND I JOINED THE CHEMICAL
CARCINOGENESIS GROUP TO STUDY
HOW CARCINOGENS DAMAGE THE DNA
AND THEN IMPORTANTLY HOW DOES
THAT DAMAGED DNA ACTUALLY BECOME
A MUTATION THAT CAN FUEL CANCER
CELL EVOLUTION.
SO FOR THOSE GRADUATE STUDENTS
AND POST-DOCS AND EARLY CAREER
TRAINEES GOING THROUGH GRADUATE
SCHOOL YOU HAVE A SMALL
CONTRIBUTION, MOST OF US, TO THE
SCIENTIFIC FIELD.
MY CONTRIBUTION WAS ACTUALLY
FOCUSSED ON MUTO GENESIS.
BACK IN THE 1980s WE STUDY
SPECIFICITY WITH TARGET REPORTER
ASSAY S THAT WE COULD LOOK AT
SEQUENCING AND THIS WAS THE
FORERUNNER OF WHAT WE NOW CALL
MUTATIONAL SIGNATURE.
STUDYING THIS IN HUMAN CELLS AND
CULTURE I STUDIED THE
SPECIFICITY I OBSERVED IN THE
HUMAN CELLS WAS VERY MUCH AKIN
TO WHAT YOU COULD OBSERVE IN
TUMORS AFTER MICE WERE INJECTED
WITH THE SAME ALKYLATING AGE
AGAINSTS BUT IF YOU FIRST
INDUCED THE IT SOS RESPONSE I
SHIFT THE SPECIFICITY AND GOT A
SPECTRUM AKIN TO WHAT I OBSERVED
IN THE HUMAN CELLS.
SO THIS REALLY FASCINATED ME.
IT CHANGED MY WAY OF THINKING
BECAUSE I THOUGHT HOW A DNA
LESION INTERACTS WITH THE DNA
POLYMERASE AND WHAT DO WE KNOW
ABOUT THIS PROCESS.
TO STUDY THIS FURTHER I JOINED
TE LAB AND FOCUSSED ON HOW DNA
POLYMERASES MAKE ERRORS EITHER
WILD TYPE DNA REPLICATED OR
DAMAGED DNA, HOW DO YOU GO FROM
A LESION TO MUTATION?
AND HOW WE KNOW DNA POLYMERASE
ERRORS ARE KEY DETERMINATES OF
THE ERRORS THAT OCCUR DURING
TUMOR CELL EF -- EVOLUTION AND
THE DNA THAT DO THIS ARE THE DNA
POLYMERASE AND WE'RE AT AN
EXCITING TIME IN THE LATE 1980s
AND WE HAD EXTRA CRYSTAL
STRUCTURES WHERE THE FIELD WAS
ADVANCING AND HAD DNA
POLYMERASES AND THIS IS A
STRUCTURE OF ONE OF THE FIRST
POLYMERASES CRYSTALLIZED WITH
THE DNA AND SUBSTRATES AT HIGH
RESOLUTION AND WE ALSO HAD
CRYSTAL STRUCTURES OF THE DNA
AND OLGA KINARD PUBLISHED AND
STUDIED HOW THE STRUCTURE OF DNA
MISPAIRS WAS DIFFERENT THAN THE
STRUCTURE OF WHAT WE KNOW IS THE
DIFFERENT PAIRS IN THE
LABORATORY I WAS ABLE TO STUDY
DNA POLYMERASE AND BECOME A BIO
CHEMIST AND I LAEARNED IT'S A
FAMILY OF ENZYMES AND WAS ABLE
TO WORK FROM HIV REVERSE
TRANSCRIPTASE AND LEARNED TIME
MANAGEMENT SKILLS BECAUSE AT THE
TIME AS THE POST-DOC MY THIRD
CHILD WAS BORN AND MY SONS ISAAC
AND IAN I HAD AS A GRADUATE
STUDENT BUT THE REAL CHALLENGE
FOR ME FOR HOW DO YOU DO
WORK-LIFE BALANCE CAME DURING MY
POST-DOC YEARS WHERE PERFORMING
THE EXPERIMENTS, WRITING THE
MANUSCRIPTS AND DOING THE
THINKING.
THIS WAS A FORMATIVE TIME FOR ME
AS A POST-DOCTORAL FELLOW.
ARMED WITH ALL THIS NEW
KNOWLEDGE THEN I WAS LOOKING FOR
FACULTY POSITIONS AND I ACTUALLY
JOINED THE FACULTY AT PENN STATE
AND JOINED THE CAMPUS OF
MEDICINE AND IN THE DEPARTMENT
OF PATHOLOGY AND THE DEPARTMENT
WAS GROWING AND A NEW DIVISION
WAS BEING EXPANDED FOR RESEARCH
AND SPECIFICALLY LOOKING FOR
PEOPLE STUDYING CANCER RESEARCH.
SO PUTTING EVERYTHING TOGETHER
FOR MY GRADUATE SCHOOL TRAINING
AND MY POST-DOCTORAL TRAINING I
WENT ON TO STUDY ONE OF THE
BIOCHEMICAL MECHANISM UNDER LIES
THE IT MUTATIONS DURING CANCER
DEVELOPMENT.
SO MY LABORATORY EXCUSE ME
PHONE.
SORRY ABOUT THAT.
MY LABORATORY HAS SEVERAL
EXPERIMENTAL APPROACHES THAT WE
USE TO STUDY THE MECHANISMS OF
MUTATION IN HUMAN CELLS DURING
CANCER DEVELOPMENT RANGE FROM
BIOCHEMISTRY THE TOPIC OF WHAT
I'LL TELL YOU ABOUT TODAY,
LOOKING AT THE RESPONSES OF
CELLS IN CULTURE TO VARIOUS
TYPES OF STRESSES AND HOW THAT
CHANGES THE PROCESS OF MUTO
GENESIS AND NOW RECENTLY USING
GENOMICS TO EXPLORE WHAT'S
HAPPENING IN HUMAN TISSUES.
THE DNA HAS TO BE COMPLETELY
DUPLICATED AND FINISHED BEFORE
CELLS DIVIDE IN CYTOKINESIS.
SO MY LAB HAS STUDIED OVER THE
YEARS POLYMERASES FROM VARIOUS
FAMILIES.
I HAVE ACCORDING TO POSTULATED
FUNCTIONS HERE.
WE'LL BE TALKING ABOUT THE
REPLICATIVE POLYMERASES AND
WE'VE DONE A LITTLE BIT OF WORK
WITH DNA REPAIR ENZYMES BUT MOST
THE WORK HAS BEEN ON WHAT I
GROUP TOGETHER AND CALL
SPECIALIZED POLYMERASES.
ENZYMES THAT HAVE AND CREATE
GENOME REPLICATION.
AND WE WANTED TO STUDY THE HUMAN
GENO
GENOME.
AS SOON AS YOU ASK THE QUESTION
WHAT IS THE SEQUENCE OF HUMAN
GENOME YOU ARE UP TOO THE
REALIZATION THAT 60% OF THE
HUMAN GENOME IS COMPRISED OF
REPETITIVE DNA SEQUENCES OF
VARIOUS TYPES.
MANY ARE FAMILIAR WITH THE
MICROSATELLITES FOUND AT
TELEMERE SEQUENCES AND FOUND
WITHIN COMMON FRAGILE TYPES AND
OTHERS CONSEQUENCES AND
IMPORTANTLY REPETITIVE SEQUENCES
ARE FOUND THROUGHOUT THE GENOME
AND CAN BE SITES OF RECURRENT
STRUCTURAL VARIATION IN CANCER
CELLS.
WHEN YOU THINK OF REPETITIVE
SEQUENCES YOU HAVE TO THINK
ABOUT THE POLYMERASES.
THEY HAVE TWO SUBSTRATES.
ONE IS THE DNA AND ONE IS THE
INCOMING DNTP.
DNA COMES IN MANY SECONDARY
STRUCTURES AS A BIO PHYSICIST
CAN BE RANDOM AND WHEN YOU BEGIN
THE SEQUENCE OF DNA IT WILL FOLD
INTO A SECONDARY STRUCTURE.
AND THEY'RE CALLED IN THE FIELD
AS NON BDNA.
DEF
AND THEY MAKE A
SOMATOMUTOGENESIS AND DISCUSSED
IN THE LITERATURE AS DIFFICULT
TO REPLICATION.
TODAY WE'LL TAKE A DEEP DIVE TOO
THE DIFFICULT TO REPLICATE
SECOND-CHANCES I'LL CALL DITORS.
IMAGINE YOU'RE A CAR AND THE DNA
POLYMERASE IS THE DRIVER AND YOU
NEED TO NAVIGATE THE TRACK THE
HUMAN GENOME.
AT TIMES YOU NEED TO DRIVE NICE
SMOOTH ROADS AND OTHER TIMES
YOU'LL ENCOUNTER OTHER OBSTACLES
SUCH AS HAIRPINS, BUMPS IN THE
ROAD WILL YOU NAVIGATE TO THE
END ON TIME AND NOT MAKE MISTAKE
TO FLIP YOUR CAR OVER AND THE
OUTCOMES OF INHIBITION AND
ERRORS ARE ACTUALLY TWO EVENTS
IMPACTFUL FOR GENOME STABILITY
AND CAN LEAD TO CHROMOSOME
BREAKAGE AND MUTATION.
TODAY LIKE I SAID WE'LL FOCUS ON
THE DETOURS AND THE DIFFICULT TO
REPLACE AND WE'LL SEE WHAT
CONSTITUTES A DETOUR AND WHAT
MAKES A SEQUENCE DIFFICULT TO
REPLACE.
OUR FOCUS WILL BE ON DNA
POLYMERASE AND SHOW YOU HOW WE
USE BIOCHEMISTRY TO GET TO THE
DETOUR.
NEXT I WANT TO FOCUS ON OUR MORE
RECENT WORK ASKING THE QUESTION
OF WHAT IS THE COMPLETE
LANDSCAPE OF DETOURS WITHIN THE
HUMAN GENOME AND FINALLY I'M
JUST GOING TO TOUCH ON A
QUESTION THAT IS A SEPARATE
PROJECT IN MY LABORATORY AND
THAT IS WHEN DO THESE DETOURS
ACTUALLY BECOME THREATS TO
GENOME STABILITY DURING CANCER
CELL EVOLUTION.
SO TO DEFINE DETOURS, WE SET OUT
MANY YEARS AGO TO TRY TO SET UP
A BIOCHEMICAL SYSTEM TO ASK WHAT
MAKES A SEQUENCE DIFFICULT TO
REPLICATION.
WE'RE FOCUSSED ON THE LOCUS TO
STUDY.
FRA 16 IS A FRAGILE SITE WITH
COMMON BREAKS FREQUENTLY SEEN IN
TUMOR CELLS AND USED THE GENOME
BROWSER TO IDENTIFY DIFFERENT
WINDOWS WITHIN THE FRA 16 LOCUS
THAT HARBOR VARIOUS TYPES OF
REPETITIVE SEQUENCES SUCH AS
QUASI-PALINDROMES.
WE NEXT CLONED THE SEQUENCES
INTO THE FACTOR SO ISOLATE
SINGLE STRAND DNA FORMS OF THE
VARIOUS DITORS.
ALL THE SEQUENCES ARE CLONED IN
THE SAME LOCATION AND HAVE A
CONSTANT UPSTREAM AND DOWN
STREAM REGION SO THE POLYMERASE
WILL TAKE A RUNNING START DOWN
THE HIGHWAY AND ENCOUNTER THE
VARIOUS DETOURS.
AS A CONTROL, WE ACTUALLY CLONED
IN A SEQUENCE FROM FRA 16D THAT
HAS AN EQUIVALENT AT CONTENT OF
76% BUT NO REPETITIVE ELEMENTS.
SO OUR ASSAY IS FAIRLY
STRAIGHTFORWARD.
IT'S A PRIMER EXTENSION EASY
WITH QUANTIFICATION AT THE
NUCLEAR STRAND LEVEL AND ALLOW
THE POLYMERASES TO SYNTHESIZE
THROUGH AND SEPARATE ALONG SIDE
THE DNA SEQUENCING LADDER TO SEE
EXACTLY AT WHAT POINT THE
PRODUCTS ARE ACCUMULATED.
WE THEN CAN QUANTITATE USING
QUANTITATION AND HOW MANY
PRODUCTS ARE ACCUMULATED WITHIN
THE DITORS SEQUENCE VERSUS HOW
MANY ARE ABLE TO SYNTHESIZE
THROUGH THE DITORS INTO THE DOWN
STREAM REGION.
THIS A QUICK SNAPSHOT OF THE
FIRST ASSAY.
THIS IS LOOKING AT THE R1 TO R5
WHICH ARE DIFFERENT REGIONS, THE
DIFFERENT WINDOWS ALONG THE FRA
16 LOCUS AND WHAT YOU CAN SEE IS
THAT THERE ARE SEVERAL REGIONS
WHERE DNA POLYMERASES ARE FAIRLY
HAPPY SYNTHESIZING THROUGH.
THIS DIFFERENT COLORED BARS ARE
INCREASING TIME OF SYNTHESIS.
HOWEVER YOU CAN SEE THAT FOR THE
TWO STRANDS THESE ARE
COMPLIMENTARY STRANDS A AND B
THERE ARE SLOW ZONES AND THE
SLOW ZONE IS WITHIN A SPECIFIC
CHROMOSOMAL REGION OF FRA 16D
AND FOR INSTANCE HARBOR TWO
REPETITIVE SEQUENCE ONE SAY LONG
A28 MICROSATELLITE AND THE OTHER
IS A QUASI-PALINDROME OF 36
SPACES.
LOOKING UP CLOSE AT THE
NUCLEOTIDE RESOLUTION, WE ARE
NOW LOOKING AT THE DNA
POLYMERASE DELTA REGION AND YOU
CAN SEE THE POLYMERASE STARTS
OUT AND REALLY HAS DIFFICULTY
SYNTHESIZING THROUGH THIS SUCH
THAT FAIRLY LITTLE PRODUCT IS
FORMED DOWNSTREAM AND THAT IS
QUANTITATED HERE ON THE RIGHT
FOR YOU.
WE BELIEVE THIS IS DUE TO DNA
BENDING AS IT SYNTHESIZES THE
SECOND STRAND.
THE TRIMER DUPLEX STEM BECOMES
BENT AND EVEN IN THE PRESENCE OF
PCNA AND RCNA CONTINUALLY
LOADING THE CLAMP YOU LOOK AT
THE LAST LANE PLUS THE
EXPERIMENT THIS IS PLUS EXCESS
DA DNA TRAP AND ALL THE PRODUCTS
THEN ARE WITHIN THIS DETOUR IN
THIS FRA 16 REGION WITH NO
PRODUCTS EXTENDED DOWN STREAM.
THIS IS ANOTHER EXAMPLE IN THE
DATA WERE PUBLISHED IN
COLLABORATION WITH WORK ON FLEX
1 WITHIN THE FRA 1 LOCUS.
I CHOSE THIS BECAUSE YOU CAN SEE
HERE THIS IS THE ENZYME AND YOU
CAN SEE THE LIGHT DEPENDENT
INCREASE IN DETERMINATION AND
THE POLYMERASE DELTA IS
STRUGGLING TO GET THROUGH THE
LONG REPEAT AND TO SUMMARIZE THE
DATA WE CAN SEE DITORS CAN BE
DEFINED BY THEIR LENGTH
DEPENDENT INHIBITION OF HUMAN
POLYMERASE DELTA.
SHORT REPEATS ARE ARE NOT A
PROBLEM, INTERRUPTED REPEATS BUT
IT'S THE LONG MACRO SATELLITES
OR THE QUASI-P PA-PALINDROMES THAT
ARE QUITE STABLE AND IDENTIFIED
A NEW ONE IN COLLABORATION
IDENTIFYING THE HEXONUCLEOTIDE
REPEAT IN A RECENT STUDY.
SO FORTUNATELY FOR THE HUMAN DNA
THERE'S HELP FOR REPLICATIVE
POLYMERASES AND OUR LAB HAS BEEN
STUDYING WHAT ENZYMES CAN HELP
AND LOOKED AT ENZYMES BAY --
BETA AND KAPPA AND WHAT ASSISTS
IN REPLICATION AND SHOW YOU NEW
DATA TODAY THAT WE'VE JUST
GOTTEN THE PAST YEAR LOOKING AT
THE EFFECTS OF RPA.
THIS IS A SNAPSHOT TAKEAWAY
PICTURE LOOKING AT THE
DIFFERENCE BETWEEN THE
REPLICATIVE ENZYMES AND THE
SPECIAL POLYMERASE.
THEY'RE INHIBITED WITHIN THE DO
TOUR.
IT'S A QUASIPALINDROME WHICH I
MENTIONED HAS A LONG STEM.
ALL THREE ARE INHIBITED AT THE
BASE OF THE STEM WHEREAS
POLYMERASES BETA AND CAN
SYNTHESIZE READILY INTO THE DOWN
STREAM REGION.
WE PUBLISHED SEVERAL PAPERS
SHOWING THEY CAN DISPLAY THROUGH
TE DITORS THAT INHIBIT DELTA AND
WANTED TO SHOW YOU DATA SHOWING
WE BELIEVE THAT THEY CAN
INTERACT AND CAN ACTUALLY BE
ENGAGED AT THE FORK.
THIS IS AND IF WE GIVE EITHER
THEY'RE VERY EFFICIENT AT TAKING
OVER THOSE TERMINATED PRODUCTS
AND COMPLETING SYNTHESIS THROUGH
THIS REGION.
WE ALSO SHOWED IN THE PAPER
THEY'RE RESISTANT TO ACETYL
CHOLINE AND WHAT WE SHOWED IS
THAT IF WE DO THE SAME REACTION
IN THE PRESENCE APHIDICOLIN IS
DEPENDENT ON BETA AND KAPPA.
I WANTED TO SHOW NEW DATA ON
DITORS REPLICATION.
I GET ASKED WHAT IS THE IMPACT
OF A SINGLE STRAND BINDING
PROTEIN ON RPA SYNTHESIS.
IT'S EXPECTED TO MELT DOWN
SECONDARY STRUCTURES AND
STIMULATE SYNTHESIS.
I HAVE A COLLABORATION WITH PENN
LAB WITH A GIFTED BIOCHEMIST AND
CHEMIST AND HE AND HIS GRADUATE
STUDENT HAVE NOW BEEN ABLE TO
RECAPITULATE A SYSTEM
BIOCHEMICALLY WHERE WE CAN
MEASURE RPA SUCCESS AND THIS
SLIDE SHOWS THEIR SYSTEM WHERE
WE CAN ACTUALLY CONFIRM THAT RPA
IS ACTIVE IN IT'S UNWINDING
ABILITY.
SO IN THIS ASSAY WHAT THEY DID
WAS TO SYNTHESIZE A DITORS
CONTAINING TEMPLATE.
THIS IS AN AT25 REPEAT THAT IS
FOUND IN FRA 3B AND SUCH THAT
WHEN THE HAIRPIN IS FORMED YOU
HAVE HIGH THREAT ACTIVITY AND
YOU CAN SEE THAT HERE
QUANTITATIVELY.
WHEN RPA IS ADDED TO THE
REACTION THE SIGNAL GOES AWAY
VERY QUICKLY AS RPA ASKS TO BIND
THE DNA AND UNWIND THE HAIRPIN
AND THIS SUBSTRATE IS ADDED IT
JUMPS OVER AND WE GET OUR FRET
ACTIVITY BACK.
THIS SYSTEM THEY WERE ABLE TO
SHOW RPA IS ACTIVE AND WE CAN
LOSE PCNA ON TO THE REACTIONS.
SO THIS DATA NOW ARE LOOKING AT
WHAT HAPPENS WHEN RPA AND PCNA
ARE LOADED ON TO A TEMPLATE.
AND THERE'S CONDITIONS THEN TO
LOOK AT SYNTHESIS OF PAL DELTA
THROUGH THE HAIRPIN REGION.
YOU CAN SEE HERE THE GEL SHOWING
POL DELTA SYNTHESIZES AND HAS A
BRIEF PAUSE AT THE HAIRPIN AND
REPLICATES THROUGH THE END
GIVING FULL LENGTH PRODUCT AND
THE DATA IS QUANTITATED HERE.
UNDER THE CONDITIONS OF EXCESS
RPA, THIS PARTICULAR DITORS IS
NOT A SIGNIFICANT BLOCK TO POL
DELTA AND WE WANT TO LOOK AT THE
EFFECTS OF RPA ON OTHER DITORS
OF OTHER SEQUENCES TO DETERMINE
WHICH DITORS RPA IS MOST ACTIVE
IN HELPING POL DELTA SYNTHESIS.
SYNTHESIZE.
FOR THE SECOND HALF OF MY TALK I
WANTED TO SHIFT GEARS TO
UNPUBLISHED DATA AND WHAT WE'VE
BEEN WORKING ON OVER THE LAST
TWO YEARS AND THAT IS TO ANSWER
THE QUESTION OF WHAT IS THE
COMPLETE LANDSCAPE OF DITORS IN
THE HUMAN GENOME AND TO ASK
WHETHER ANY OF THESE SEQUENCES
CAN ACT AS COMPLETE ROAD BLOCKS
OR OBSTRUCTIONS TO DNA
POLYMERASES.
THIS PROJECT BEGAN AS AN
INTEGRATIVE APPROACH WITH TWO
OTHER COLLEAGUES AT PENN STATE
UNIVERSITY AND ONE WHO IS I
STATISTICAL GENOMICS GURU AND
TOGETHER THE THREE OF US HAVE
ACTUALLY BEEN CROSS-TRAINING
MANY GRADUATE STUDENTS AND
POST-DOCS TO ASK THE QUESTION OF
HOW REPETITIVE SEQUENCES ARE
MAINTAINED IN PRIMATE GENOMES
AND IN THE HUMAN GENOME.
SO WE TOOK THIS CROSS-TRAINING
COLLABORATIVE APPROACH AND
APPLIED IT TO ANSWERING THE
QUESTION OF WHAT ARE THE ALL
DITORS WITHIN THE HUMAN GENOME.
THIS WORK WAS PUBLISHED A FEW
YEARS AGO AND IT IS THE BRAIN
CHILE OF A FORMER GRADUATE
STUDENT AND A POST-DOC AND HE
HAD AN IDEA I DIDN'T THINK WOULD
WORK AND READ THERE WAS A VALUE
IT GIVES EVERY TIME IT
INCORPORATES A BASE AND HIS IDEA
IS ISN'T THIS A KINETIC VALUE
FOR A DNA POLYMERASE GOING
THROUGH THE DIFFERENT VALUES OF
THE GENOME AND I SAID YES, THIS
WOULD BE A K POL VALUE IF YOU
WANTED TO EXTRAPOLATE IT SO HE
TOOK THE DATA AND THEN ANNOTATED
ALL THE NON-BDNA SEQUENCES AND
SHE APPLIED THE TESTING TO
COMPUTE HE'S DISTRIBUTION TO
ANALYZE STATISTICAL
DISTRIBUTIONS AND THEIR
DIFFERENCES AND THEY FOUND THE
SHAPES DO DIFFER DEPENDING ON
THE SEQUENCE BEING EXAMINED AND
ALL THE G4 MOTIFS THEY EXAMINED
HAD SIGNIFICANTLY HIGHER IPDs
COMPARED TO CONTROLLED WINDOWS
AND IDENTIFY ANOTHER NON-B
MOTIFS WITH ALTERED
POLYMERIZATION KINETICS
INCLUDING THOSE IDENTIFIED IN
THE STUDY SUCH AS A-PHASE
REPEAT.
THEY ALSO SHOWED THE SEQUENCING
ERROR RATES DURING THE SYNTHESIS
ARE ARE ACTUALLY INCREASED FOR
G4 REGIONS VERSUS MOTIF FREE
WINDOWS.
THEN THEY LOOKED AT THE
G4LOCI AND FOUND THE SNIPS
COMPARED TO THE CONTROL WINDOWS
OF THE GENOME.
I WANT YOU TO REMEMBER THE
CONCEPT OF STABLE G4s.
THE QUADRANT ANALYSIS IS
ACTUALLY BUILT ON -- IT TAKES
INTO ACCOUNT G4 SEQ DATA AS WELL
AS PARTICULAR MOTIF.
THE QUADRANT SCORES ARE GIVEN
DIFFERENT VALUES AND A SCORE
GREATER THAN 19 WAS CONSIDERED
BY THE WRITERS OF THE ALGORITHM
TO BE STABLE TO THE GENOME.
IF YOU LOOKED AT STABLE G4s
THERE'S A HIGHER DIFFERENTIAL
BETWEEN SNIP FREQUENCY AND
CONTROL WINDOWS.
SO MY LAB NOW HAS STEPPED INTO
THIS PROJECT AND TAKEN OVER TO
ASK WHETHER G4s CAN BE DITORS
AND WHETHER THEY CAN AFFECT
FIDELITY AND ARE ALSO ASKING
WHETHER G4 SEQUENCES INHIBIT THE
REPLICATIVE ENZYMES.
THESE ARE THE DATA I'D LIKE TO
SHARE WITH YOU TODAY.
THESE ARE UNPUBLISHED DATA.
PURSE I WANT TO TALK ABOUT THE
POLYMERASE FIDELITY AND THE WORK
OF MY CURRENT GRADUATE STUDENT
WHO TOOK OUR EXISTING POLYMERASE
ASSAY AND WORKED VERY HARD TO
LEARN HOW TO PURIFY G4
CONTAINING SUBSTRATES AND CREATE
THE MOLECULES FOR POLYMERASE GAP
FILLING REACTION S AND CONTAIN
THE G4 MOTIF AND THEN TAKE THE
PRODUCTS AND CREATE THEM INTO
THE MUTANTS AND THEN INDEPENDENT
MUTANTS ARE SEQUENCES TO
DETERMINE THE SPECIFICITY.
SO MARY ELIZABETH CHOSE TWO G4
MOTIF.
ONE IS WITHIN THE L4 LOCUS.
AND THIS HAS A TM OF AROUND 76
DEGREES.
SHE ALSO ANALYZED THE ONE WITH
FIVE G TRACKS.
THIS IS A WELL CHARACTERIZED G4
AND IT FORMS A PARALLEL
STRUCTURE AND IS MORE STABLE.
THEN MARY ELIZABETH CREATED A
MUTANT FORM THAT ONLY CONTAINS
FOUR 4 TRACTS AND LOSING THAT
FIFTH TRACK DID NOT CHANGE ITS
STABILITY.
SO USING THIS ASSAY MARY
ELIZABETH WAS ABLE TO SHOW
FIDELITY WAS MAINTAINED DURING
DAP -- DNA SYNTHESIS AND WE
HAVE POL KAPPA AND POL BETA IS
THE LEAST ACCURATE.
I HAVE A CLOSE UP AND
QUANTITATED WHETHER THE ERRORS
ARE OCCURRING WITHIN THE G4
MOTIF VERSUS THE SURROUNDING
INTERNAL CONTROL.
AND YOU CAN SEE THAT FOR THE
DIFFERENT POLYMERASES AND G4
MOTIFS THEY'RE NOT A HOT SPOT OF
INCREASED POLYMERASE ERRORS.
HOWEVER, THAT DOESN'T MEAN TO
SAY G4s ARE NOT AFFECTING THE
POLYMERASES.
WHAT MARY ELIZABETH OBSERVED IS
SHE HAS AN INCREASED FREQUENCY
IN OUR GENE IN THE THREE PRIME
END SO WHAT YOU'RE LOOKING AT
HERE IS A CLOSE UP OF A
MUTATIONAL SPECTRUM OF THE
TARGET.
SHOWN IN RED ARE THE SEQUENCES
OF THE G4 MOTIF SO THE G4 WINDOW
IN THE BRACKETS.
SHE THEN CREATED WINDOWS OF THE
EQUIVALENT SIZE TO THE G4 MOTIF.
I THINK VISUALLY YOU CAN SEE AS
THE G4 BECOMES MORE STABLE THIS
IS ADA HAS AN INCREASED
FREQUENCY OF ERRORS WITHIN THIS
IT THREE-PRONG BLANKING REGION.
THE IT DATA IS QUANTITATED FOR
YOU HERE AND ANALYZED
STATISTICALLY AND FOR POL ADA
AND KAPPA WE SEE THE CHANGE IN
DISTRIBUTION.
AGAIN AN EXCESS OF ERRORS IN THE
FLANKING REGION FOR THE TWO G4
MOTIFS THAT FORM STABLE PARALLEL
STRUCTURES.
WE ALSO SAW A GREAT BREADTH OF
POLYMERASE ERRORS FOR ANYONE WHO
STUDIES THIS IS AN ENZYME THAT
MOSTLY MAKES SUBSTITUTIONS.
IT'S KIND OF BORING BUT WE
STARTED TO SEE QUITE A RANGE
THEN OF DIFFERENT TYPES OF
MUTATIONS WITHIN THE G4 MOTIF
AND MANY OF THESE INCLUDED BOTH
THE WHAT WE WOULD EXPECT OF THE
G TRACTS AND ALSO THE LOOPS.
HAS TO ARE LARGER INSERTIONS AND
WE'RE SEEING GREAT INCREASE IN
THE FREQUENCY OF LARGER
DELETIONS.
BUT POL DELTA ALSO MAKES AN
INTERESTING NEW CLASS OF
MUTATIONS WHICH I HAVE NOT
OBSERVED PREVIOUSLY IN MY
STUDIES IN WHICH THERE'S
INSERTIONS HERE IN THE UPPER
CASE IN THE MIDDLE OF THE G4
TRACK SO THESE INSERTION
MUTATIONS ACTUALLY SERVE TO
EXPAND THE G4 MOTIF.
THIS IS IN THE PARALLEL
STRUCTURE G4s.
WE MENTIONED DELETION MUTATION.
THIS IS NOT AN ERROR WE NORMALLY
SEE WITH POL ADA BUT WHEN
SYNTHESIZING THROUGH THE STABLE
G4s WE SEE LARGE DELETION
BASICALLY REMOVE THE BLANKING
SEQUENCE OF THE TK GENE.
WHAT DOES THIS MEAN
BIOLOGICALLY?
TO GET TO THE QUESTIONS OF THE
BIOLOGICAL CONSEQUENCES OF
POLYMERASE ERRORS WE AGAIN
WORKED WITH OUR COLLABORATORS
AND HE TOOK ALL OF THE MUE TAPTS
AND ANALYZED THEM FOR THE
QUADRANT SCORE SO AGAIN QUADRANT
IS A MACHINE ALGORITHM BASED ON
DATA FROM G4 SEQ AND
INCORPORATES THE FEATURES AND WE
MRO
PLOT THE CHANGE YOU SEE THE
ALTERED DISTRIBUTION AND SOME OF
THE MUTANTS HAVE ELIMINATED THE
ABILITY TO FORM A G4.
THESE MUTANTS THAT ELIMINATE THE
G4 POTENTIAL INCLUDE
SUBSTITUTION OR FRIENDSHIPS THAT
CHANGE THE G TRACK AND THE LOOPS
OR THE LARGE DELETIONS THAT
REMOVE THE G4.
SO WHAT ABOUT THE OTHER HALF?
TWO G4s INHIBIT HUMAN POLYMERASE
DELTA.
THIS IS A PROJECT OF SUSAN HILE
AND STUDIED THIS FOR L1 SEQUENCE
AS WELL AS ANOTHER MOTIF BASED
ON THE QUADRANT SCORE THESE ARE
NOT EXPECTED TO BE VERY STABLE
STRUCTURES AND THIS IS CONFIRMED
ANTI-PARALLEL STRUCTURES AND
KNOW G4 MOTIF WITHIN THE L1
SEQUENCE THIS HAS A FAIRLY HIGH
QUADRANT SCORE EXPECTED TO BE
STABLE AND INDEED LOOKING AT THE
THERMO STABILITY IN OUR REACTION
BUFFERS AND CIRCULAR
CONFIGURATION IS EXPECTED TO BE
A STABLE STRUCTURE AND FORM
INSTRA STRAND STRUCTURES.
THE QUESTION IS HOW DO THEY
IMPACT THE DNA POLYMERASES?
THE SYSTEM IS THIS SAME.
AN EX TENSE SYSTEM WHERE WE LOOK
AT THE G4s WITHIN THE SINGLE
STRANDED VECTORS AND LOOKING AT
THE UNSTABLE AND STABLE
STRUCTURES AND IF YOU LOOK AT
THE L1 SEQUENCE SUSAN ALSO
ANALYZED POLYMERASE PROGRESSION
THROUGH THIS SEQUENCE OF THE G
RICH AND C RICH STRANDS.
WHAT I HOPE YOU CAN SEE IS THIS
IT VERY PRONOUNCED PAUSING OF
DELTA AT THE BASE OF THE G4 AND
THE DATA ARE QUANTITATED HERE
WHERE ALL THE G4s DO HAVE
SOMEWHAT INCREASED TERMINATION
BUT THE STABLE G4 IS THE HIGHEST
AMOUNT AND THAT IS NOT SEEN ON
THE COMPLIMENTARY STRAND.
WE'RE ASKING DO THE TWO
POLYMERASES AT THE REPLICATION
FOR DELTA AND EPSILON REMAIN
THROUGH A G4 CONSEQUENCE.
THIS IS A CLOSE UP OF PAPER FROM
THE COLLABORATORS AND THE G4
MOTIF HAVE A LARGE DISTRIBUTION
OF INCREASED INTERPULSE DURATION
STRANDS AND THE IT OTHER DOESN'T
SHOW THAT TYPE OF DISTRIBUTION.
WE SAW WHERE THE G STRAND HAS
MORE INHIBITION THAN THE C
STRAND.
TO LOOK AT THIS QUESTION SUSAN
HAS DEVELOPED A SYSTEM LOOKING
AT A VARIETY OF G4 MOTIF.
SO IN ADDITION TO THE THREE WE
LOCKED AT WE ALSO ASKED OUR
GENOMICS COLLABORATORS WHAT ARE
THE MOST ABUN DAND STABLE MOTIFS
PREDICTED STABLE MOTIFS IN THE
HUMAN GENOME.
THE IT DATA ARE SHOWN ON THE
RIGHT.
THESE ARE THE NUMBER OF G4
MOTIFS AN YOU SEE GREAT
ENRICHMENT WITHIN THE TWO MOBILE
ELEMENTS AND THE L1PA ELEMENTS.
SO WE CREATED ANOTHER MOTIF THAT
IS ONE THAT CORRESPONDS TO THE
ELEMENT THEY'RE PROMINENT IN
THE HUMAN GENOME AND WANTED TO
LOOK AT G4s AND WE CHOSE ONE OF
THE OVER SEQUENCES THAT WERE
DESCRIBED SEVERAL YEARS AGO.
THIS WAS ONE OF THOSE SHOWN TO
BE FUNCTIONALLY WORK AS AN
ORIGIN IN THE SEQUENCE.
AND WE COLLABORATED WITH A
FORMER POST-DOC AND WE WANT IT
LOCK AT THE REPLICATIVE ENZYMES
AND ASK THE QUESTION WHAT IS TO
THE SYNTHESIS THROUGH THE G4
MOTIF AND WE'LL QUANTIFY
PRODUCTS BECAUSE THESE ARE GOING
TO COMPLIP ALL THE WAY AROUND
OUR CIRCLE.
THESE ARE SMALL CIRCLE.
WE'RE GOING TO ASK HOW MANY
PRODUCTS ARE INHIBITED AND HOW
CAN SYNTHESIZE PAST THE G4 AND
WE HAVE TWO REACTIONS ONE FOR
THE G RICH AND ONE FOR THE C
RICH STRAND AND WHAT WE'RE DOING
IS WE'RE SETTING UP A SYSTEM
MIMIC WHAT IS GOING ON WHERE THE
POL DELTA WOULD BE ON ONE STRAND
THE G RICH STRAND AND POL
EPSILON IS FOUND ON THE C RICH
STRAND.
YOU CAN SEE IN EITHER
ORIENTATION, BOTH THE ENZYMES
ARE GREATLY INHIBITED BY THIS
PARTICULAR SEQUENCE I'M SHOWING
HERE.
THIS EX TENTS PRECEDING THE
CONSEQUENCE.
WE BELIEVE WE'RE GETTING
STRUCTURE FORM IN THE
DOUBLE-STRANDED TEMPLATE AND
BECAUSE WE'RE LOOKING AT THE
MULTI SUB UNIT ENZYMES WE'RE
SEEING INHIBITION OF THE
POLYMERASES AS THEY APPROACH THE
G4.
THESE IS PRELIMINARY DATA SO FAR
JUST TO GIVE AN EXAMPLE OF WHAT
WE'RE DEVELOPING.
AND DELTA GETS THE C RICH STRAND
AND YOU CAN SEE THE
DETERMINATION THE PARTITIONING
VALUE IS QUITE BIASSED TOWARDS
TERMINATION ON THE G RICH
STRAND.
WE SEE THE SAME SITUATION IN THE
OPPOSITE SO IF THE FORK IS GOING
IN THE OTHER DIRECTION NOW DELTA
GETS THE G RICH STRAND AND ALSO
HAS A HIGHER PARTITIONING VALUE
COMPARED TO THE RANDOM CONTROL.
WE WANT TO TAKE THE DATA ONE
STEP FURTHER TO DEVELOP A
REPLICATION UNCOUPLING INDEX TO
GET AT THE QUESTION OF THIS
DISCONNECT OF POLYMERASES ON THE
TWO STRANDS AS THEY'RE
SYNTHESIZING THROUGH A G4.
WE TAKE THE PARTITIONING VALUE
FOR EPSILON COMPARED TO DELTA ON
THE C RICH STRAND AND THAT WOULD
BE OUR LEADING STRAND UNCOUPLING
INDEX.
AND YOU CAN SEE THAT THE G4
MOTIF FROM IS QUITE BIAS.
POL EPSILON HAS QUITE A BIT AND
WE CAN COMPARE THE POLYMERASE
REPLICATION THROUGH WHAT SHOULD
BE CONSIDERED A NORMAL TEM
POLITICAL REGION AND YOU CAN SEE
THAT BOTH OF THE IT NOBLE
ELEMENTS AND VA AND L1, G4s HAVE
INCREASED UNCOUPLING INDEX.
SO WHAT I'VE TOLD YOU FOR OUR
RECENT RESEARCH LOOKING AT G4s
IN THE GENOME HOW THE
POLYMERASES NAVIGATE THROUGH,
WE'VE LOOKED AT MUTATIONS AND
FOUND SO FAR THE HUMAN
POLYMERASE ERROR FREQUENCY
WITHIN THE G4 WILL NOT GREATLY
ELEVATED.
THE G4s THEMSELVES DON'T HAVE A
HAD THE SPOT TO THE POLYMERASE
BUT THE PRESENCE OF THE G4
INCREASES ERRORS IN THE BLANKING
REGION.
THE ERRORS WITHIN THE G4 ARE
EXPECTED TO INCREASE AND
DECREASE STABILITY OVER
EVOLUTIONARY TIME THERE SHOULD
BE A MAINTENANCE OF HE IT G4s
MONTREAL
MAINTENANCE AND IN THE GENOME
AND WHAT IS OBSERVED IN THE GERM
LINE ARE PROBABLY DUE TO
INHIBITING THE PAIR OR
INCREASING THE IT DNA DAMAGE.
FOR REPLICATION WE OBSERVED SOME
BUT NOT ALL G4s DO REPLICATIVE
DNA SYNTHESIS AND HAVE PAUSING
BY THE DELTA AND THE ENZYMES ON
THE G RICH STRAND AND THAT
UNCOUPLING INDEX IS GREATEST
WHEN THE G4s ARE IN THE LEADING
STRAND CONFIRMATION MEANING
EPSILON GETS THE G RICH STRAND.
WHAT WE WANT TO DO NOW OF COURSE
IS LOOK AT HOW OTHER REPLICATION
PROTEINS CAN ASSIST IN
SYNTHESIZING THROUGH THESE G4s
THAT ARE EXPECTED TO BE
INHIBITORY.
SO IN THE LAST TWO MINUTES, WHAT
I WANTED TO TELL YOU WAS JUST TO
GIVE YOU AN IDEA OF THE OTHER
PROJECTS IN MY LABORATORY AND
THAT THEY ARE SUBCULTURE BASED
AND THEY'RE TRYING TO ASK THE
QUESTION WHEN DO THESE DITORS
BECOME THREATS TO HUMAN GENOME
STABILITY.
THE REPLICATION STRESS WE'RE
FOCUSSING ON ARE THINGS THAT WE
KNOW WILL IMPACT DNA POLYMERASES
SUCH AS THE DNTP LEVELS THAT MAY
BE LOWERED BY REDUCTASE.
I MENTIONED EXHAUSTION IS
SOMETHING THEY'RE IT INTERESTED
IN LOCKING AT THE EFFECT ON
DITORS.
MY LAB STUDIED DEFICIENCIES OF
POLYMERASES AND I WANTED TO SHOW
YOU ONE LAST BIT OF DATA
ONCAGENE DATA AND I WANTED TO
SHARE IT WITH YOU AND THEY'VE
BEEN RECENTLY PUBLISHED AND
THESE ARE THE DATA OF GRADUATE
STUDENT WHO JUST DEFENDED LAST
YE
YEAR AND THIS WAS A MUTANT FORM
OF H RAS AND DID IT IN
FIBROBLASTS.
SO OUR EXPECTATION IS ONCA GENE
EXPRESSION WAS UP REGULATE DNA
POLYMERASES BECAUSE PREVIOUSLY
MY LAB HAD SHOWN IN A
PUBLICATION THAT IF YOU TREAT
CELLS WITH AN HU YOU GET UP
REGULATION AT THE PROTEIN LEVEL
OF DNA POLYMERASE BETA THIS IS
AN INDEPENDENT MECHANISM AND
IT'S FOR CHROMATIN FRACTION AND
WHAT HE SAW WAS THE OPPOSITE.
I REPEATED SOME OF THESE
EXPERIMENTS AND IT'S QUITE
SURPRISING TO ALL OF US IN THAT
WHAT WE OBSERVED WAS THE DNA
POLYMERASES ARE RAPIDLY DEPLETED
UPON ONCA GENE REPLICATION AND
WE RECENTLY PUBLISHED THIS AND
YOU CAN SEE THIS RAPID DOWN
REGULATION OF A DA, CKAPPA AND
THE POL D3 FOR THOSE WHO STUDY
BREAK INDUCED REPAIR AND
POLYMERASE SELFA.
I HAVE TAKE THEN THE CULTURES
OUT AND IS THE CELLS ARE RESTED
AND THEY DID NOT TOTAL LLY SENESCE
YOU CAN CONTINUE THESE FOR WEEKS
AND TO ME IT'S QUITE PROVOCATIVE
THERE COULD BE A WINDOW OF
VULNERABILITY AS FAR AS GENOME
INSTABILITY DUE TO THE ALTERED
DNA REPLICATION PROGRAM IN
RESPONSE TO ONCA GENE
ACTIVATION.
THANK YOU ALL VERY MUCH FOR YOUR
ATTENTION.
I THINK I'VE MENTIONED EVERYONE
ON THE PENN STATE TEAM WHO
HELPED WITH THE STUDY AND I HAVE
MY FUNDING SOURCES.
THANK YOU AGAIN AND I'LL BE
HAPPY TO TAKE ANY QUESTIONS.
>> THANK YOU, KRISTIN.
VERY WONDERFUL TALK.
I APPRECIATE YOUR GOING TO THE
BACKGROUND.
THERE'S A FEW QUESTIONS HERE.
ONE FROM THE UP BOX DOES DNA
REPAIRS REPEAT AND IS IT STALLED
AND ARE TRIPPLEXES MUTOGENIC.
>> THAT'S A GREAT QUESTION.
THANK YOU.
WE HAVE LOOKED AT TRIPLEX DNAs.
THIS WOULD BE IN POLY PURIN
TRACKS WHEN THE MICROSATELLITE
TRACKS BECOME SUCH THE SINGLE
STRAND SNAPS BACK AND THE SINGLE
STRAND SNAPS BACK TO PERFORM THE
TRIPLEX.
THEY DO BECOME DETOURS WHEN
THEY'RE LONG ENOUGH.
SOMETHING WE'RE VERY INTERESTED
IN BECAUSE WE ARE NOW STUDYING
THE GGGA SEQUENCES THAT ARE TOO
SHORT TO FORM A TRIPLEX SO WE
MONITORED THEM BY CHANGING PH
AND USING THIS AND THE QUESTION
WHETHER THEY'RE HOT SPOTS FOR
MUTOGENESIS I WOULD DIRECT YOU
TO -- WE HAVE NOT STUDIED THE
MUTOGENESIS AS WELL AS SHE HAS.
>> THANKS, KRISTIN, VERY NICE
TALK.
ANY ON THE INHIBITED POL DELTA?
OUR HYPOTHESIS WOULD BE WE HAVE
NOT BEEN ABLE TO SET UP OUR DUEL
POLYMERASE REACTION WITH PCNA
WITH POL IOATA IT'S A
BIOCHEMIST'S CHALLENGE TO GET
THE BUFFER SYSTEM TO GET THE
ACTIVITIES HAPPENING TOGETHER TO
CO -- DO THE STUDIES.
WE'RE INTERESTED IN THOSE TYPES
OF ANALYSES.
>> IN THE HSV2K ASSAY HAVE YOU
LOOKED AT THE IN VITRO REACTION
AFFECTS FREQUENCIES AND
SPECTRUM?
>> WE HAVE NOT DONE THAT TYPE OF
ANALYSIS YET.
>> THOSE INVOLVED IN THE
POLYMERASE DELTA
BETA, KPV AND KPD ARE YOU
CONSIDERED THEM AND HAVE YOU
LOOKED AT THEM?
>> I WANT TO LOOK AT MORE HE LA
CASES.
TO DATE WE'VE ONLY LOOKED AT ONE
IN COLLABORATION WITH A LAB.
I'M INTERESTED IN MORE OF THE
RG4 PROJECT, FOR EXAMPLE.
I ACTUALLY HADN'T CONSIDERED
WHAT YOU SUGGEST ED IT HAS AN
ACTIVE ROLE AND I NEVER
CONSIDERED THAT SO THAT WOULD BE
SOMETHING I'D BE INTERESTED IN
DOING, YES.
>> WE FOUND IT INVOLVED IN
TRANSCRIPTION AS WELL REPAIR AND
PATIENTS HAVE MANY DIFFERENT
FEATURES AND IT'S HARD TO
EXPLAIN THEM ALL.
THE NEUROLOGIC SYSTEM AND
DYSREGULATION.
AND YOU'VE BEEN TALKING ABOUT
POL ADA.
SO ANDREAS, WOW, FEW MORE
QUESTIONS.
FOR REPLICATION WAS THERE ANY
OTHER DIFFERENCE BETWEEN THE POL
DELTA AND EPSILON CASES AND
LOOKING AT MAGNESIUM
CONCENTRATION.
>> EXCELLENT QUESTION.
SUSAN HILE WORKED HARD ON MAKING
THE REACTION CONDITIONS
IDENTICAL FOR THE DELTA AND EP
-- EPSILON AND ALL REACTIONS
CONTAINED AND PCNA AND RSC WE
KNOW THE RFC WAS ACTIVE IN
LOADING THE PCNA.
WE TOOK CONDITIONS ON THE G RICH
AND C RICH STRAND AND USED
CONDITIONS WHERE THE ENZYMES ALL
GOT UP TO THE STARTING LINE SO
THEY'RE RUNNING START WITH WERE
THE SAME, REV YOUR ENGINES, ALL
THE MOLECULES ARE AT THE
STARTING LINE AND GO THROUGH THE
G4 AND THAT'S HOW WE CAME UP
WITH OUR BIOCHEMICAL APPROACH TO
ASK THIS STRAND DIFFERENTIAL.
WE HAVE NOT YET ATTEMPTED TO ADD
ANY MORE PROTEINS TO THE
REACTION BUT IT'S SOMETHING WE
DEFINITELY HAVE ON THE BOOKS TO
DO.
>> THANK YOU.
OKAY, TELOMERES ARE SEQUENCES
WOULD EXPECT HIGH MUTATION
FREQUENCIES AND IS THERE DATA ON
THIS?
>> I WOULD DIRECT YOU TO PATTY
OPRE SCA'S LAB AND SOMETHING
THEY'RE LOOKING AT AND I'M NOT
IN THE TELOMERE BIOLOGY FIELD.
WE CHOSE OUR G4s TO BE THOSE
THAT ARE MOST ABUNDANT IN THE
GENOME THE NOBLE ELEMENTS IN THE
OTHER G4s AND HAVE NOT LOOKED AT
TELOMERIC REPEATS IN MY LAB.
>> AND HAVE YOU TESTED
POLYMERASE LEVELS IN AN ONCA
GENE EXPERIMENTAL SYSTEM?
>> WE HAVE.
THAT WAS PART OF MY STUDENT'S
THESIS.
THIS IS SPECIFIC -- WELL, WE
LOOKED AT TWO.
ATRAS AND CMEK AND IT DOES NOT
INDUCE THE SAME POLYMERASE
DEPLETION AND WE CAN USE A
PATHWAY BUT I TOLD MY STUDENTS
THIS WILL IT BE ANOTHER 10 YEARS
FIGURE OUT THE MECHANISM AND HE
NEEDED TO GRADUATE SO WE HAVE
THE OTHER ONCA GENES IN THE
FREEZER BUT WE DIDN'T DO THEM
YET.
GREAT QUESTION AND SOMETHING WE
ABSOLUTELY WANT TO TRY TO FIGURE
OUT WHAT'S GOING ON WITH THE
SYSTEM.
>> WELL, THANK YOU SO MUCH.
AS IT BEEN A WONDERFUL SEMINAR.
AND HAVE A HAPPY HOLIDAY AND
NICE BEGINNING OF WINTER NOW.
THIS THE WINTER SOLSTICE AND WE
HAVE PEOPLE CLAPPING HERE.
>> THANK YOU FOR THE INVITATION.
IT WAS MY PLEASURE AND THANK YOU
ALL FOR LISTENING AND HAVE A
HAPPY AND HEALTHY NEW YEAR.