>>WELCOME TO THE DNA
REPAIR INTEREST GROUP CONFERENCE
SERIES.
THIS IS THE LAST 1 OF THE SEASON
AND WE WILL BE TAKING A BREAK IN
JULIE AND --JULY AND AUGUST AND
PICKING UP IN SEPTEMBER.
THOSE OF YOU WHO WOULD LIKE TO
BE UPDATED ON THE CONFERENCENESS
AND ALSO INFORMATION ABOUT DNA
REPAIR MEETINGS AND POST DOCS,
PLEASE JOIN THE LISTSERV, THE
DNA REPAIR INTEREST GROUP
LISTSERV, AND SEND IT TO ME.
WE'RE HAPPY TO HAVE
DR. TOM KUNKEL SPEAK TO US, HE
IS AT THE NIEHS WHERE HE'S BEEN
FOR 40 YEARS IN 1982 BUT HE
RECEIVED HIS EDUCATION AT THOMAS
MOORE COLLEGE IN KENTUCKY.
HE HAS A BACHELOR'S AND HE GREW
UP ON A FARM IN KENTUCKY TO HELP
US AND HE MOVED TO CINCINNATI
UNIVERSITY
WHERE HE GOT A MASTERS IN
CELL BIOLOGY AND A Ph.D. IN
DEVELOPMENTAL BIOLOGY AT THE
UNIVERSITY OF CINCINNATI.
HE THEN BEGAN POST DOCTORAL
TRAINING WITH DR. LARRY LOWE WHO
IS REALLY WELL KNOWN FOR HIS
WORK IN MUTE O GENESIS,
APPARENTLIA LARRY WAS INITIALLY
IN PENNSYLVANIA BUT THEN WENT TO
THE UNIVERSITY OF WASHINGTON TBH
SEATTLE WHERE HE IS STILL.
DR. KUNKEL STARTED WITH HIM IN
1978 AND THEN STAYED WITH HIM
UNTIL HE CAME TO NIH AND NIEHS
AND RESEARCH TRIANGLE NORTH
CAROLINA, OVER THE YEARS HE'S
MOVED UP, HE WILL GRADUATE, HE
BECAME THE CHIEF OF THE
LABORATORY OF STRUCTURAL BIOLOGY
AND ALSO DIRECTOR OF THE
ENVIRONMENTAL BIOLOGY PROGRAM AT
NIEHS, SINCE 2011 THOUGH, HE HAS
A SPECIAL TITLE OF AN NIH
DISTINGUISHED INVESTIGATOR.
AND THOUGH HE IS VERY PROLIFIC
IN RESEARCH HE'S RECEIVED MANY
HONORS INCLUDING AN HONORARY
DOCTOR, DOCTORAL DIPLOMA FROM
[INDISCERNIBLE] UNIVERSITY IN
SWEDEN.
AND HE HAS BEEN ELECTED TO THE
AMERICAN ACADEMY OF ARTS AND
SCIENCES HAS BEEN ON NUMEROUS
EDITORIAL BOARDS, GAVE MANY
LECTURES AND KEY NOTE SPEECHES
AND PUBLISHED MORE THAN 400
PAPERS, RESEARCH PAPERS IN THE
BEST JOURNAL IN THE COUNTRY, OR
IN THE WORLD.
DR. BOER HAS A LITTLE MORE TO
SAY,.
>> YEAH, JUST TO CHIME IN HERE,
TOM, IT'S A PLEASURE TO HAVE YOU
AS A TRUE LEADER IN THE FIELD OF
DNA REPAIR, MUTOGENESIS AND HAS
BEEN A LEADER FOR MANY YEARS,
I'VE HEARD HIM ON MANY
OCCASIONS, VERY OFTEN AS A KEY
NOTE TALKING ABOUT MUTOGENESIS,
FED ILLEGALSITY OF POLYMERASES,
ROLE OF RNA AND IT'S REALLY
GREAT TO HAVE YOU TOM AND I
WANTED TO ALSO ADD THAT THIS
SAYS YOU HAVE ACHIEVED NUMEROUS
AWARDS, YOU'VE GONE THROUGH ALL
LEVELS OF NIH AND NOW
DISTINGUISHED INVESTIGATOR AND
RECEIVED MANY AWARDS.
I'M VERY IMPRESSED WITH THE
PRINCESS TAKAMATSU, THAT WE
TALKED ABOUT ONCE ON A TRIP WHEN
WE WERE TOGETHER IN JAPAN,
THAT'S 1 VERY PRESTIGIOUS AWAR.
IT'S VERY GREAT TO HAVE YOU AND
SEE YOU.
>> TOM WILL BE SPEAKING ABOUT A
NUMBER OF TOPICS, INITIALLY
ADVERTISE WITH EXTRINSIC PROOF
BREEDING OF THE DNA REPLICATION
ERRORS BUT HE'S EXPANDED TO IN
GENERAL EUKARYOTIC DNA
REPLICATION FIDELITY BY THE BETA
FAMILY OF DNA POLYMERASES.
SO, IF YOU HAVE ANY QUESTIONS,
PLEASE PUT IT IN THE CHAT BOX
IT'LL BE DIRECTED TO ME AND
DR. BOER AND WE WILL READ THE
QUESTIONS AT THE END.
THANK YOU.
OKAY, TAKE IT AWAY, TOM.
>> WELL, I WANT TO THANK KEN AND
WILL FOR THE KIND INVITATION TO
SPEAK HERE.
THIS IS A REAL PLEASURE AND I'M
LOOKING IF ORDER TO INTERACTING
WITH ANY OF YOU IF YOU HAVE ANY
QUESTIONS.
FIRST THING I WANT TO SAY IS
THIS TALK AND ALL MY TALKS FOR
THE INDEFINITE FUTURE ARE GOING
TO BE IN MEMORY OF SAM WILSON,
SAM WAS A VERY CLOSE FRIEND OF
MINE AND 1 OF THE BEST
SCIENTISTS I KNOW AND HE DIED
LAST YEAR AND WE ALL MISS HIM
VERY MUCH, MANY OF THE PEOPLE IN
THIS AUDIENCE, THAT'S PRETTY
MUCH ALL I WANT TO SAY EXCEPT
THAT HE REALLY WAS A FINE--HE
AND I SHARED BIRTHDAYS ABOUT 10
YEARS APART OR CLOSE TO SHARING
BIRTHDAYS AND EVERY TIME I HAD A
CAREER DECISION OF ANY SORT TO
MAKE, I ALWAYS CONSULTED WITH
SAM AND HE WAS ALWAYS VERY
GENEROUS IN HIS MENTORING ADVICE
IN ADDITION TO PROVIDING AN
EXCELLENT EXAMPLE FOR ME OF HOW
TO DO QUALITY SCIENCE SO I HAVE
FOND MEMORIES OF SAM AND I HOPE
YOU DO TOO.
SO THE TITLE OF MY TALK IS
EUKARYOTIC DNA REPLICATION
FIDELITY BY THE B-FAMILY DNA
POLYMERASES.
THESE ARE THE PERSONS CURRENTLY
IN MY GROUP.
MY GROUP IS CURRENTLY AS SMALL
AS IT'S BEEN FOR THE LAST 35
YEARS, PARTLY DUE TO THE COVID
PANDEMIC.
BUT THE REAL POINT HERE IS IN
ADDITION TO THANKING ALL OF THEM
FOR THEIR FINE WORK IN MY GROUP.
I WOULD LIKE YOU TO TAKE NOTE
THAT I HAVE POST DOCTORAL
POSITIONS CURRENTLY AVAILABLE IN
MY LAB, IF YOU'RE INTERESTED,
ACCEPTED ME AN E-MAIL AND WE CAN
CHATS.
THIS IS A TIMELINE FOR LIFE ON
EARTH STARTING WITH FORMATION,
THE MOON, THROUGH THE PRESENT
DAY, AND ON THE UPPER RIGHT YOU
CAN SEE VISIBLE LIFE, IT HAS
ONLY BEEN AROUND FOR ABOUT A
HALF OF A BILLION YEARS OF THE
4.5 BILLION YEAR LIFE SPAN ON
THIS OR SPAN ON THIS DIAGRAM AND
HUMANS HAVE ONLY BEEN AROUND FOR
ABOUT 200,000 YEARS, SO, 1 OF
THE INTERESTING THINGS FROM MY
PERSPECTIVE ABOUT THE WORK WE DO
APPROXIMATE IS THE EVOLUTIONARY
PERSPECTIVE AND 1 WAY TO SAY
THAT IS JUST TO POSE A SIMPLE
QUESTION.
I LOVE SCIENCE FICTION MOVIES, I
LOVE THE MIEWRY JUREACIC PARK
AND WHEN I SAW THOSE DINOSAURS
WALKING AROUND, I WONDERED HOW
THEY WERE REPLICATING THEIR DNA
AND THAT WAS MANY MILLIONS OF
YEARS AGO.
DO THEY HAVE THE SAME ENZYMES
THAT I'M GOING TO TALK ABOUT IS
INTERESTING TO ME.
IN ADDITION, FROM THE STANDPOINT
OF HOW WE'RE GOING TO
INVESTIGATE THAT, HERE'S A SLIDE
ON PROTEIN DYNAMICS AND
MACROMOLECULAR MOTIONS THAT CAN
OCCUR OVER TIME FRAMES OF 10 TO
THE FOURTH SECONDS ON THE LEFT
TO 10 TO THE MINUS 142ndS ON
THE RIGHT HAND SIDE OF THE RED
BAR.
AND WHAT IS STUDIED ARE LISTED
ABOUT THAT RED BAR AND HOW
THEY'RE STUDIED IS LISTED BELOW
THAT RED BAR AND THE POINT OF
THESE 2 SLIDES IS TO SAY THAT
OUR UNDERSTANDING OF DNA
REPLICATION FIDELITY SPANS RANGE
FROM 2 ANG STROMs, TO 2-METERS
THE SIZE OF SOMEONE
APPROXIMATELY AND 10 TO THE 14
FRAMES MINUS TO 14-SECONDS
GREATER THAN 14 YEARS, SO THAT'S
A HUGE SPECTRUM OF SPACE AND
TIME AND TO STUDY THAT THE
TECHNOLOGIES THAT ARE CURRENTLY
IN USE ARE STRUCTURAL BIOLOGY,
BIOCHEMISTRY, GENETICS AND
GENOMICS.
AND I WILL ILLUSTRATE CERTAIN
POINTS WITH EACH OF THOSE 4
APPROACHES TO UNDERSTANDING DNA
REPLICATION FIDELITY SO HERE ARE
THE SUBUNITS OF THE DNA
POLYMERASES IN 4 FAMILIES.
THERE ARE 17 OF THEM THAT ARE
NOW KNOWN, MOST OF THEM ARE
SHOWN HERE AND WHAT I WANT TO
FOCUS THIS TALK ON ARE DNA
POLYMERASES OUT FOR DELTA AND
EPSILON WHICH ARE IN THE
B-FAMILY.
HERE THEY ARE WITH A SUMMARY OF
SOME OF THEIR FUNCTIONS AND
PROPERTIES, THEY'RE ALL
MULTISUBUNIT DNA POLYMERASES,
THEY ARE HIGHLY CONSERVED FROM
BUDDING YEAST, WHAT MY TALK WILL
BE ABOUT THROUGH FISSION YEAST
AND HUMANS AND THEY HAVE
ACTIVITIES IN ADDITION TO THE
DNA POLYMERASE ITSELF THAT ARE
RELEVANT TO REPLICATION.
THEY'RE FIDELITY FROM WORK THAT
ME AND OTHER VS DONE FOR AT 10
TO THE MINUS 4 AND BETTER FAN
THAT PRACTICES PROEPSILON, AND
DEDICATION IS WHAT I WILL TALK
ABOUT HERE.
SO NUCLEIIC DNA DEPENDS ON THE
CONCENTRATIONS OF THE DNTs,
AND THE RMTPs AND THAT
INCLIEWRDS ABSOLUTE
CONCENTRATIONS AND RELATIVE
CONCENTRATIONS AND DEPENDS ON
THE DNA POLYMERASES FOR CORRECT
PROPERLY ALIGNED DTNP AND THE
CORRECT SUGAR MOIETY, AND PROOF
ON REPLICATION WHICH WE NOW
BELIEVES OCCURS IN 2 FLAVORS, 1
IS EXTRINSIC PROOF READING AND
THE OTHER IS EXTRINSIC PROOF
READERRING AND IT DEPENDS ON
REPAIR OF ERRORS AFTER
REPLICATION BY DNA MISMATCHED
REPAIR AND NUCLEOTIDE EXCISION
REPAIR.
HERE ARE THE 4 PROCESSES THAT I
WOULD LIKE TO CONSIDER IN THIS
BRIEF TALK, 1 IS CHAIN
ELONGATION ON A TEMPLATE THAT
OCCURS ABOUT 6 BILLION TIMES FOR
EVERY REPLICATION CYCLE, THAT'S
HOW MANY NUCLEOTIDES ARE IN THE
HUMAN GENOME.
OKAZAKI FRAGMENT MATURATION
WHICH I WILL MENTION BREFLY
OCCURS 25 MILLION TIMES
PERNUCLEAR GENOME.
RIBONUCLEOTIDE EXCISION PAIRS,
RIBONUCLEOTIDES INCORPORATED
DURING REPLICATION OCCURS
3 MILLION PER GENOME PER CYCLE
AND MISMARCHED REPAIR OCCURS A
HUNDRED OR MORE TIMES PER
REPLICATION CYCLE.
AND THE REASONS WHY I AM
INTERESTED ARE UNDERSTANDING THE
RESPONSIBLE MECHANISMS FOR THESE
PROCESSES AND CONSISTENT WITH
THE INTERESTS OF THE NATIONAL
INSTITUTES OF HEALTH, THEIR
CONNECTIONS TO HUMAN HEALTH AND
AS I MENTIONED AT THE
BEGENERATEDDING, THE
EVOLUTIONARY CONSERVATION OF THE
ENZYMES AND PROCESSES THAT OCCUR
FOR EACH OF THESE 4 STEPS.
SO WHAT I'M GOING TO DO NOW IS
WALK THROUGH A TINY BIT OF
INFORMATION THAT WE ACCUMULATED
OVER 40 YEARS I'VE BEEN AT THE
NIEHS, ON EACH OF THESE 4
PROCESSES.
ONE WAY TO DO THIS IS TO STUDY
MUTATOR DNA POLYMERASES, SO
HERE'S AN EXAMPLE OF THE POLE
DELTA BINDING POCKET OF THIS
CRYSTAL ENZYME STRUCTURE
PUBLISHED BY THE CANNED WHAT LAB
SEVERAL YEARS AGO AND IT SHOWS
THE ACTIVEVIATE WITH A NEWLY
FORMING BASE PAIR AND RESIDUES
SURROUNDING THAT NEWLY FORMING
BASE PAIR INCLUDING Y2613 NPOL
DELTA WHICH IMMEDIATELY AT THE
BOTTOM IS HIGHLY CONSERVED IN
ALL 4 EUKARYOTIC DNA POLYMERASES
AND IMMEDIATELY ADJAC TONIGHT
THAT, THE RESIDUE THAT WE'VE
CHANGED WHICH IS EITHER A LUCINE
OR POLE ALPHA OR POLE DELTA OR A
METRICS THININE, CONSERVED
METHIONINE IN POLE EPSILONE.
SO WE'VE CHANGED THOSE AMINO
ACIDS TO OTHER AMINO ACIDS AND
AMONG OTHER THINGS WE
DEMONSTRATED THEY'RE ALL MUTATOR
POLYMERASES AND HERE IS A WHOLE
GENOME MUTATION RATE ANALYSIS OF
8 DIP EMPLOYED BUDDING YEAST
STRAIN, THEY ARE ALL CONTAINED
EITHER THE WILD-TYPE AND/OR THE
MUTATOR OF EPSILONE, ALPHA AND
DELTA AND THEY CONTAIN
MISMATCHED REPAIR EITHER
PROEFFICIENT AND THIS IS A THIS
IS A BRIEF SUMMARY OF WHAT WE
LEARNED ABOUT PUBLICATION IN
WHICH WE PERFORMED WHOLE GENOME
SEQUENCING OF EACH OF THESE 8
STRAINS, PASSAGE THROUGH ABOUT
900 GENERATIONS OF GROWTH ON
PETRI DISHES AND HERE'S SOME OF
THE RESULTS, THIS IS THE NUMBER
OF DISTRIBUTION OF SINGLE BASED
CHANGES THAT WE DISCOVERED IN
THESE 8 STRAINS, THE LEGEND IS
ON THE UPPER LEFT AND EACH OF
THESE COLORED MARKS THERE
REPRESENT WHERE MUTATIONS WERE
FOUND IN THE 16 CHROMOSOMES OF
THESE YEAST STRAINS.
AND THEN AFTER 9 GENERATIONS
LOOKED AT EACH OF THESE IN BLUE
AS WHOLE GENOMES THAT WE SEEK
DATA.
WE KNOW FROM THESE TYPES OF
SAMPLES WE KNOW THAT LEUCINE
L-THE IPADDA 9 AND DELTA
INVITHOE AS CERTAIN SPECIFICITY
INCLUDING THE G OPPOSITE T,
MISPAIRS, 28 MORE FREQUENTLY
THAN C OPPOSITE A AS DESCRIBED
IN THE UPPER LEFT OF THIS SLIDE.
AND IF YOU MAP THOSE ON THE
CHROMOSOME, YOU CAN FIND THAT
THE PREVENTIVE RENTERAL
PERFORMANCE OF G-OPPOSITE T
OCCURS JUST TO THE RIGHT OF
THORIGEINSOT LEFT-HAND SIDE, AND
JUST TO THE LEFT OF ORIGINS ON
THE RIGHT HAND SIDE WHICH MEANS
THAT THEY'RE PRIMARILY LAGS
STRAND DNA REPLICATION ERRORS
AND THEIR DISTRIBUTION ACROSS A
CHROMOSOME IS IN RED AND BLUE
X-DIAGRAM ON THE LOWER LEFT AND
IF YOU CONSIDER THIS KIND OF
INFORMATION FOR EACH OF THE 12
MISPAIRS THAT CAN FORM, AND FOR
EACH OF THE 3 MAJOR REPLICATIVE
DNA POLYMERASES, ALPHA DELTA AND
EPSILONE, WE COME UP WITH A
MODEL THAT WE BELIEVE IS
CURRENTLY GOING ON IN YEAST
CELLS WHICH IS THAT POLE EPSILON
IS THE MAJOR LEADING STRAND
REPLICATION, AND THE POLE DELTA
DOES THE BULK OF CHAIN
ELONGATION OVER THE LAGGING
STRAND.
YOU CAN TEST THIS HYPOTHESIS
FURTHER USING A PROCEDURE CALLED
HYDROLYTIC DNA IN-SEQUENCING
WHICH WE DEPRIVATIONED BACK IN
PUBLISHED IN 2015, THAT'S THE
PROCEDURE ITSELF, BUT THIS TELLS
YOU WHERE EACH OF THE 3
POLYMERASES INCORPORATE
RIBONUCLEOTIDES INTO THE
CHROMOSOMES IN NUCLEIC
CHROMOSOMES IN BUDDING YEAST AND
THIS IS 1 EXAMPLE OF FROM 2015
WHERE IN THE MIDDLE OF THIS
SLIDE, THE RED AND GREEN--WHERE
THE RED AND BLUE SWITCH AND IF
WE LOOK AT THE PATTERNS OF WHERE
THESE 3 MUTANT DNA POLYMERASES
INCORPORATE RIBONUCLEOTIDES AND
WE GET A PICTURE THAT INDICATES
THE ROLES OF REPLICATION AND
IDENTIFIES REPLICATION ORIGINS,
SO WHERE WE ARE RIGHT NOW, IS
SHOWN IN THIS SLIDE, THE CURRENT
MODEL IS SHOWN IN THE TOP PANEL
A WHICH IS--SHOWS THAT POLE
EPSILONE, IS THE MAJOR LAGGING
STRAND REP LIAISON CASE, POLE
DALTA IS THE MAJOR LAGGING
STRAND, POLE EPSILONE IS THE
MAJOR LEADING STRAND, SHOWS
ORIGINS OF REPLICATION WHERE
POLL ALPHA IS DOING MOST OF THE
INITIATION OF OKSCRKS AKI
FRAGMENTS ON THE LAST STRAND.
HOWEVER, WE SINCE PUBLISHED THAT
DURING INITIATION OR
REPLICATION, POLL DELTA,
SYNTHESIZES POLL ALPHA AND DELTA
SIPGHTICIZES THESE ORIGINS AND
REPLICATION AND THEN WHEN POLL
DELTA BUMPS INTO THE POLL
EPSILON, INACTIVE BUT NAILED IT,
IT'S A COLLISION BETWEEN THOSE 2
ENZYMES ALLOWING POLL EPSILONE,
TO INITIATE LEADING STRAND
REPLICATION, BUT WITHOUT DENYING
THE FACT THAT THE WHOLE ALPHA
AND DELTA SYNTHESIZED BOTH THE
LEADING AND THE LAGGING STRAND
WITH ORIGINS OF APPLICATIONS
LIAISONPLICATION DURING
APPLICATION AND THEN PANEL C
ILLUSTRATES WHAT WE THINK IS
GOING ON WHEN 2 REPLICATION
FORKS COLLIDE WITH 1 ANOTHER
FROM OPPOSITE ORIGINS AND THERE,
EPSILONE, HANDINGS THE DNA BACK
TO POLE DELTA WHICH DOES LEADING
AND LAGGING, SO OUR OVERALL
CURRENT WORKING HIPOLGT SIS IS
THAT POLE EPSILONE IS THE
LEADING DELTA REP LIAISON CASE
BUT ALPHA AND DELTA MAY BE OF
THE LEADING STRAND AT ORIGINS AT
TERM NATIONS BEING A PREDOMINANT
LEADING STRANDS OF THESE
ENZYMES.
SO REPLICATION IS COMPLICATED
BUT FOR THE VAST MAJORITY OF
REPLICATION, AT LEAST OF THE
YEAST CHROMOSOMES WE BELIEVE
POLE EPSILOINY IS THE LEADING
CASE AND DELTA AND ALPHA ARE THE
SYNTHESIZING, THESE FRAGMENTS.
IN 1989 AND 1990 THERE WERE 22
PUBLICATIONS, 1 BY KATHRYN AND 1
BY FRED ANDULARLY, WHICH
PROVIDED BIOCHEMICAL EVIDENCE
SUGGESTING THAT AFTER A
POLYMERASE GENERATES A MISMATCH,
THAT MISMATCH CAN BE REMOVED BY
AN EXONUCLEASE IN A SEPARATE
PROTEIN.
AND THAT'S WHAT WE NOW MY LAB
REFERRED TO AS EXTRINSIC PROOF
READING WHERE THE POLYMERASE
THAT MAY HAVE BEEN THE MISTAKE
IS NOT THE SAME PROTEIN THAT
PROOF READS THIS THING.
SO INTRINSIC PROOF READING OF
MISMATCH IS MADE AND IS THEN
MOVED WITHOUT INTERVENING
DISASSOCIATION OR THE
POLARIZEDDER MERACE, WHEREAS
EXTRENSIC PROOF IF A MISMATCH IS
MADE AND THAT DISASSOCIATES AND
THEN SOME ENZYME THAT THEN BINDS
TO THE MISMATCH IN THE
EXONUCLEASE ACTIVE SITE AND
REMOVES THE MISMATCH.
SO WE WERE INTERESTED IN TESTING
THIS HYPOTHESIS AND WE'VE DONE
SO NOW, AND WE NOW HAVE EVIDENCE
FOR EXTRINSIC PROOF READING OF
REPLICATION ERRORS, BY POLL
DELTA.
ONE PUBLICATION WAS PUBLISHED IN
2006 AND IT SHOWS A SYNERGISTIC
INCREASE IN THE MUTATION RATE
WHEN WE HAVE A MUTATOR
DERIVATIVE OF POLL ALPHA ON THE
LAGS STRAND INITIATING OKSAKI
FRAGMENTS AND WE COMBINE THAT
WITH A PROOF READING DEFICIENT
DERIVATIVE OF POLE DELTA, 35 DB
AND WHEN YOU COME BIEN THOSE 2,
YOU GET A SYNERGISTIC INCREASE
IN MUTATION RATE THAT'S
ILLUSTRATED BY THAT HIGH GRADE
BAR ON THE LEFT AND IN 2021,
DR. ZHU LED A STUDY IN MY LAB
WHICH SHOWS A SYNERGISTIC
INCREASE ONLY PRIMARILY FOR POLE
EPSILONE ERRORS SO THE DATA IN
THIS ARE CONSISTENT WITH
HYPOTHESIS THAT POLE DELTA CAN
EXTRENSICICALLY DO THE
REPLICATION ERRORS IN VIVO, AND
AS YOU CAN SEE, SO FAR IN THE
UPPER LEFT, POLL 2-4
EXODEFICIENT ENZYME WHICH IS A
PROEPSILONE POLYMERASE CANNOT
EXTRINSICALLY PROOF READ ERRORS
MADE BY POLE.
BY POL-ALPHA SO SO FAR WE HAVE
EXTRINSIC READING BY POL DELTA
BUT NO EVIDENCE THAT POL E OR
POL A CAN CONTRIBUTE TO THE SAME
PROCESS.
SO THIS IS A SUMMARY OF WHAT WE
LEARNED SO FAR, EXTRINSIC POL
READING, CORRECTS MISMATCHES
MADE BY ALL 3 REP LIAISON CASES,
IT'S EFFICIENT, IT'S INDEPENDENT
OF POLYMERASE ACTIVITY OF POL
DELTA, IT'S LARGELY INDEPENDENT
OF DNA MISMATCH REPAIR, IT'S
SPECIFICITY DIFFERS FROM
INTRINSIC PROOF READING.
IT BALANCES LEADING AND LAGGING
STRAND REPLICATION FIDELITY AND
WE BELIEVE IT'S RELEVANT TO THE
ORIGINS OF CANCER AND TO
EVOLUTION AND IF YOU'RE
INTERESTED IN THESE PROCESSES WE
HAVE WRITTEN A LITTLE MINIREVIEW
OF THIS PROCESS WHICH WILL BE
PUBLISHED IN A FEW WEEKS IN THE
JOURNAL OF DNA REPAIR.
THIS IS THE SECOND SUBJECT OR
OKSAKI FRAME MATURATION, THIS
SLIDE IS SIMPLY PUT UP THERE TO
TELL YOU THAT OKSAKI FRAGMENT
MATURATION WHICH IS TD REMOVE
WILL OF RIBOTIDES INCORPORATE
BIDE POL ALPHA AND THEIR
REPLACEMENT BY POL DELTA WITH
DNA, FOLLOWED BY LIGATION TO
FORM A CONTINUOUS LAGGING
STRAND, THAT PROCESS IS VERY
COMPLICATED AND CAN BE A SHORT
PATCH PATHWAYOT LEFT OR A LONG
PATCH PATHWAY ON THE RIGHT THAT
CAN TAKE CARE OF THE LONG PATCH
AND RETURN IT TO THE PATHWAY.
THIS IS A SHORT PATCH PROCESS
BUT ON THE LEFT-HAND SIDE YOU
CAN SEE THAT WHEN WHEN A SHORT
PATCH IS THERE, FLAP ENDOCLUE
CLE ACE BINDS AND EXCISES THAT
SHORT PATCH.
AFTER SYNTHESIS BY POL DELTA TO
CREATE THAT FLAP.
AND THEN POL, THE FLAP
ENDONUCLEACE DISASSOCIATES AND
DNA LIGASE SEALS IT TO GIVE A
CONTINUOUS LAGGING STRAND
TEMPLATE.
THAT'S THE PROCESS THAT COPIES
THAT HAPPENS ROUGHLY 25 MILLION
TIMES PER EUKARYOTIC NUCLEAR
GENOME AND IT'S BEEN
DISPLACEMENT SYNTHESIS AS YOU
CAN SEE, SO WE'VE BEEN VERY
INTERESTED IN ADDRESSING WHETHER
THE FIDELITY OF THAT REACTION
DIFFERS FROM THE FIDELITY OF
MOST OF REPLICATION WHICH
REPRESENTS MOST OF THIS ON AN
OPEN TEMPLATE.
WELL 1 WAY RECENTLY THAT WE'VE
GOTTEN INVOLVED IN THAT, COMES
FROM WORK DONE IMMEDIATELY NEXT
DOOR TO US BY SCOTT WILLIAMS LAB
WHO PUBLISHED THE HIGH
RESOLUTION STRUCTURE OF DNA
LIGASE 1, BOUND TO DNA AND WHAT
THEY DISCOVER ON THE UPPER RIGHT
OF THIS SLIDE IS THAT IN
ADDITION TO THE CATALYTIC
MAGNESIUM AT THE POLYMERASE OR
AT THE LIGASE ACTIVE SITE,
THERE'S A SECOND MAGNESIUM ION
BOUND 3 NUCLEOTIDES UP STREAM OF
WHERE THE LIGATION HAS TO OCCUR
AND IF STRUCTURE IS SHOWN IN THE
MIDDLE OF THIS SLIDE, THAT LED
JESS WILLIAMS WHO IS SCOTT
WILLIAMS WIFE BUT WORKS IN MY
LAB TO ADDRESS THE QUESTION OR
DNA LIGASE FIDELITY IS IMPORTANT
FOR GENOME STABILITY AND THE
APPROACH IS TO USE A BUDDING
YEAST STRAIN AND TEST THE MUTE O
GENIC CONSEQUENCES OF EXPRESSING
A LIGASE MUTANT IN THAT STRAIN
WHICH GETS RID OF THOSE HIGH
FIDELITY MAGNESIUM BINDING SITE
AND THIS IS WHAT JUST FOUND THAT
THE SPECTRUM OF MUTANTS IN THAT
LIGASE MUTANT ARE DOMINANTLY
CHARACTERIZED BY 1 NUCLEOTIDE
EDITIONS IN SHORT MONONUCLEOTIDE
ROUNDS.
THOSE ARE THOSE RED CLOSED
TRIANGLES, YOU CAN SEE SEVERAL
HOT SPOTS FOR THOSE EVENTS
OCCURRING IN THE EURO 3 GENE AND
WE BELIEVE THEY ARE REPLICATION
ERRORS IN THE OGASAKI, LIGASE IN
THE MUSEUM TABT AND WE GOT
INTERESTED IN WHETHER THEY'RE
SUBJECTED TO DNA MISMATCH REPAIR
WHICH CAN BE MY MUTE S ALPHA OR
MUTE S-BETA DEPENDENT REPAIR OR
BY FLAP PROCESSING DURING
OKASAKI, AND SO WE TESTED THIS
IN FLAP ENDONUCLEACE AND BOTH OF
THOSE PROCESSES DO AFFECT
REPLICATION FIDELITY, AND THEY
ARE SPECIFIC FOR CREATING A
LARGE INCREASE IN MUTE O
GENESIS, FOR PLUS 1 BASE FRAME
SHIFTS IN SHORT HOMONUCLEOTIDE
RUNS, SO HERE IS THE CURRENT
WORKING MODEL IN THE LAB WHERE
ON THE UPPER LEFT IS STRAND
DISPLACEMENT SYNTHESIS TO CREATE
THE FLAP, FOLLOWED BY A STRAND
SLIPPAGE EVENT TO CREATE AN
EXTRA HELICAL C-RESIDUE AND THEN
FOLLOWING THAT UPON OR ONCE THAT
OCCURS, YOU HAVE EXTRA C THAT
CAN BE INCORPORATED AND THEN THE
FLAP ENDONUCLEACE CLEAVES THE
FLAP AND THEN LIGASE IS SUPPOSED
TO LIGATE THIS INTERMEDIATE TO
CONTAIN A CONTINUOUS LEADING
STRAND BUT ON THE LOWER LEFT,
YOU CAN SEE IF THE WILD-TYPE REP
LIAISON CASE ENCOUNTERS THAT,
CAN YOU HAVE A BOARD OF LIGATION
AND END PROCESSING OR PROOF
READING TO REMOVE THAT EXTRA
NUCLEOTIDE BUT IF THE MUTANT
FORM OF LIGASE IS THERE, THIS
EE TO MUTE ANT EA, YOU GET
INSERTIONS INSERTED BY INSERTING
THE NEXT CORRECT C AND THEN MUTE
O GENIC LIGATION.
SO WE'RE VERY INTERESTED IN THE
PROCESS, AS 1 SIGNATURE OF
OKASAKI MATURATION AND WHAT I
CAN SAY IS HAD --WHAT WE'RE DOIG
NOW IS TO INVESTIGATE THAT WHOLE
PROCESS BY WHOLE GENOME
SEQUENCING TO EXAMINE THAT
CHARACTERISTIC ERROR OR
SIGNATURE PLUS 1 FRAME SHIFTS
ACROSS THE ENTIRE NUCLEAR
GENOME.
I'LL LET YOU KNOW WHAT HAPPENS,
SOMEDAY SOON.
AND THEN HERE'S 1 OF THE MORE
INTERESTING SLIDES, I WILL SHOW
YOU REPRESENTS UNPUBLISHED
OBSERVATIONS SO DON'T TAKE THIS
TOO SERIOUSLY UNTIL WE HAVE TIME
TO DO MORE WORK ON IT, BUT THIS
IS THE EURO3 MUTANT SPECTRA FOR
AN ACTIVE SITE MUTATION IN
LIGASE 1, THAT'S THE MOST MIXED
COMBINATION OF ERRORS CREATED
DURING OKASAKI MATURATION THAT
I'VE EVER SEEN.
IT INCLUDES ALL KINDS OF
MUTATIONS, PLUS 1S AND MINUS 1S,
IN RUNS, NONE RUNS AND THEN THE
BIG RED DRI ANGLES ARE INSERTION
OF OF LARGER NUMBERS OF
NUCLEOTIDES AND WHEN JESS AND I
HAVE BEEN STUDYING THIS SPECTRUM
FOR A COUPLE OF WEEKS NOW, AND
WE BELIEVE IT INDICATES AT LEAST
5 DIFFERENT MECHANISMS BY WHICH
1 CAN GET A MUTATOR PHENOTYPE BY
HAVING AN ACTIVE SITE POINT
MUTATION IN LIGASE 1.
SO THIS IS WORK THAT WE'RE
WORKING ON NOW TO BEGIN TO
INVESTIGATE THOSE MECHANISMS AND
WE WILL FOLLOW THIS SLIDE UP
WITH PUBLICATIONS, SOMETIMES IN
THE NEXT YEAR OR 2 INCLUDING
WHOLE GENOME SEQUENCING TO SEE
IF THIS KIND OF SPECIFICITY,
BROAD SPECIFICITY IS ACROSS THE
ENTIRE NUCLEAR GENOME, I SHOULD
MENTION THE EURO-3 GENE IS ABOUT
110,000th OF THE ENTIRE LENGTH
OF THE 16 CHROMOSOMES IN YEAST,
SO THERE'S A LOT OF
OPPORTUNITIES TO INVESTIGATE, A
LOT OF PROCESSES RNTION THAT
COULD BE UNIQUE TO OKASAKI
FRAGMENT MATURATION AND SINCE
THAT INVOLVES STRAND
DISPLACEMENT SYNTHESIS, WE ARE
INTERESTED IN KNOWING IF PLUS 1
ERRORS ARE COMMON TO STRAND
DISPLACEMENT SYNTHESIS REACTIONS
BY ANY OF THE OTHER 16 HUMAN DNA
POLYMERASES BESIDES
[INDISCERNIBLE].
IN FACT, WE HAVEN'T YET
DEMONSTRATED THAT THOSE PLUS 1S
ARE COMMON TO STRAND
DISPLACEMENT BY POL DELTA PER
SE, THAT'S JUST THE WORKING
HYPOTHESIS AT THE MOMENT.
HERE ARE RIBONUCLEOTIDES, ARE
THEY INCORPORATED DURING
REPLICATION?
THIS IS FOR AN INCORRECT BASE.
ON THE UPPER LEFT, IS THE SUGAR
AND THAT CAN ATTACK THE SUGAR
PHOSPHATE BACKBONE IN IT'S
PRESENT IN DNA AND DO CLEAVE AND
CREATE A NIK WITH UNLIGATABLE
DNA ENDS WHICH HAVE TO BE
PROCESSED.
ON THE RIGHT IS HOW POLYMERASES,
DNA POLYMERASES EXCLUDE
RIBONUCLEOTIDES AND ON THE
BOTTOM IS CONCENTRATIONS OF THE
DNTPs AND THE RNAs IN YEAST
MEASURED BY ANDRE CHAVEZ OUR
COLLABORATOR IN THE UNIVERSITY
AND YOU CAN SEE THAT THE
CONCENTRATION OF THE
DEOXYNUCLEOTIDE TRI POS FATES IS
MUCH LOWER THAN THE
CONCENTRATION OF THE RNTPs, BY
DIFFERENCES OF 194 OR 36 FOLD,
ET CETERA.
SO THERE'S A LOT MORE RNTPs
CONSIST WENT THEIR CRITICAL ROLE
IN TRANSCRIPTION, BUT DOES THAT
INCREASE ALSO DNA POLYMERASES TO
INCORPORATE RIBONUCLEOTIDES
BECAUSE IT'S--THEY'RE PRESENT IN
EXCESS OVER DNTPs, THAT IS
INCORPORATION ASSAY DONE INVITRO
WITH POLs ALPHA, DELTA AND
EPSILONE, AND THEY INDICATE
RIBONUCLEOTIDES AS INDICATED BY
THESE LINES WHERE THESE ARE
INCORPORATED BY THE 3 DNA
POLYMERASES AND IF 1 ASKS HOW
CELLS THERE IS A CLEAR ESTIMATE
OF A NUMBER OF RESIDUES
GENERATED PERGENOME, NUCLEAR
GENOME IN EUKARYOTES FOR A WHOLE
VARIETY OF LESIONS THAT HAVE
BEEN STUDIED FOR MORE THAN 50
YEARS AND IF WE ASK HOW MANY
RIBONUCLEOTIDES AREN'T
INCORPORATED, WE CAN GIVE EACH
OF THESE KINDS OF LESIONS
STUDIES FROM THE LAST 50 YEARS,
LITTLE CIRCLES WITH INCREASING
SIZE REPRESENTING INCREASING
NUMBER AND THEN WE CAN SHOW HOW
MANY RIBONUCLEOTIDES ARE
INCORPORATED AND YOU CAN SEE,
RIBONUCLEOTIDES ARE THE MOST
ABUNDANT NONNORMAL--THE
NUCLEOTIDE TRI PHOSPHATE
INCORPORATED DURING REPLICATION
SO THIS GOT US VERY INTERESTED
IN THIS PROCESS, AND THERE'S A
SUMMARY OF WHAT JESS WILLIAMS
AND I PUBLISHED IN THE ANNUAL
REVIEWS OF BIOCHEMISTRY THEY
THINK SHOULD BE OUT NOW,
SUPPOSED TO COME OUT ONLINE IN
MAY, AND IT HAS BOTH THE AMOUNT
OF RIBONUCLEOTIDES INCORPORATED
ON THE LEFT, THE DNA
REPLICATIONS THAT DO THAT CAN
PROOF READ, OR REMARE THOSE
RIBONUCLEOTIDES BY RNA AGE TO
DEPENDENT RIBONUCLEOTIDE
EXCISION REPAIR OR BY
TOPO-ISOMER ACE REMOVAL AND THEN
THERE'S EVEN MORE THAT'S PUTS
INNING OKAZAKI AND WE ARE
BEGINNING TO STUDY THAT PROCESS
AND ON THE RIGHT ARE THEINDS
OF EEIVETS THAT THOSE
RIBONUCLEOTIDES CAN HAVE IN
CELLS INCLUDING REPLICATION,
STRESS AND GENOME INSTABILITY
WITH THE ACKNOWLEDGMENT THAT
SOME OF THOSE EFFECTS COULD BE
RELATED TO OTHER ROLES FOR RNA
SAGE 2 FOR EXAMPLE IN RESOLVING
ALL OF THOSE, SO IF YOU'RE
INTERESTED IN THIS SUBJECT, YOU
CAN READ THAT IN THE REVIEWS
ARTICLE AS A WAY TO GET STARTED
ON THAT.
AND HERE IS EUKARYOTIC MISMATCH
REPAIR, I DON'T HAVE A CLOCK IN
FRONT OF ME, HOW AM I DOING ON
TIME?
>> YOU'RE DOING FINE, YOU STILL
HAVE 15 MINUTES OR SO.
>> OKAY, THAT'S PERFECT.
THIS IS EUKARYOTIC MISMATCH DNA
REPAIR AS WE INFER FROM A LOT OF
LITERATURE, OCCURS IN YEAST OR
HUMAN CELLS.
IT'S RECOGNITION AT THE TOP OF A
MISMATCH LEFT BEHIND BY THE
REPLICATION FORK BY MUTASE ALPHA
FOR EXAMPLE, AND THEN MUTATE
ALPHA LEADS A SEPARATE
HETERODIMER COMPRISED OF PMS 2
EXPW MLH 1 AND THEN THERE'S THE
INVOLVEMENT OF PC NA THAT
ALLOWED THEM IN THE DNA STRAND
AND THIS ALLOWS MISMATCH REMOVAL
BY ANY OF SEVERAL PROCESSES,
FOLLOWED BY DNA MISMATCHES ONCE
IT'S GONE AND THEN LIGATION TO
RESTORE GENOME INTEGRITY AND
THAT'S JUST A VERY QUICK DIRTY
REVIEW OF DNA MISMATCH REPAIR.
WHAT WE'VE BEEN DOING IS 1 SMALL
SUBSET OF THE KIND OF WORK THAT
MANY INVESTIGATORS AROUND THE
WORLD HAVE BEEN DOING, AND HERE
IS 1 EXAMPLE OF 1 OF THE UTCOMES
WE PUBLISHED LAST YEAR.
SCOTT [ [INDISCERNIBLE] IN MY LB
AND I LOOKED AT SEQUENCING A
VARIETY OF OF SPECIES SHOWNOT
LEFT-HAND SIDE AND ON THE
EXTREME RIGHT IS THE EFFICIENCY
OF MISMATCH REPAIR OF ERRORS
MADE IN THOSE ORGANISMS BY THE
REPLICATION MACHINERY AND YOU
CAN SEE THAT THE MISMATCH REPAIR
EFFICIENCY IS ESTIMATED TO BE
ROUGHLY THE SAME IN ALL THESE
ORGANISMS, WE FIND THAT
INTERESTING RELATIVE TO
UNDERSTANDING THE MECHANISMS OF
DNA MISMATCH REPAIR, 1 OF THE
WAYS THAT WE'RE INVESTIGATING
THAT S&P MAY BE THAT MISMATCH IT
ACTS IN THE DIRECTED MANNER
POSSIBLY BECAUSE OF A SPECIAL
RELATION TO THE REPLICATION
COMPLEX THAT WAS FIRST PROPOSED
BY WAGNER AND [INSCERNIBLE] IN
1966 AND ON THE LOWER LEFT, CAN
YOU SEE THERE MAY BE SOME
SPECIAL RELATIONSHIP BETWEEN
WHEN IT HOW REPLICATION ERRORS
ARE MADE BY THE REPLICATIONS AND
HOW THEY'RE REPAIRED BY THE
MISMATCH REPAIR MACHINERY.
AND SOME OF THE PROCESSES THAT
WE'RE INVESTIGATING INCLUDE
MISMATCHED REPAIR EFFICIENCY
POLYMERASE THAT MAKES THE ERROR,
THE MISMATCH REPAIR SUBWAY THAT
OPERATING, IS MUTE S ALPHA OR
MUTE S BETA, 2 DIFFERENT
HETERODIMERS, MISMATCHED
COMPOSITION, WHETHER THE ERROR
IS A LEADING STRAND OR LAGGING
STRAND REPLICATION ERROR, THE
SEQUENCE CONTEXT REPLICATION
TIMING, CHROMOSOMAL LOCATION AND
CHROMATIN STAFF, SO I WILL SHOW
YOU JUST VERY QUICKLY A COUPLE
OF UNPUBLISHED OBSERVATIONS THAT
WERE CURRENTLY WORKING HARD ON
IT.
ONE IS WE KNOW FROM LITERATURE
THAT'S BEEN PUBLISHED INCLUDING
EVIDENCE BY PAUL MODRICH WHO WON
THE NOBEL PRIZE IN 2015 FOR HIS
WORK ON THIS MISMATCH REPAIR,
THAT THERE IS A REQUIREMENT FOR
PC NA IN DNA MISMATCH REPAIR,
AND WE PUBLISHED IN 1996 THAT
THAT REQUIREMENT IS AT A STEP
PRECEDING DNA RESENTH SIS.
SO THERE ON THE LEFT IS A LITTLE
CARTOON OF MISMATCH REPAIR
PROTEINS AND ON THE EXTREME LEFT
OF MSH3 THE END TERMINUS AND YOU
CAN FIND WHAT'S CALLED A PC NA
BINDING MOTIF, AND IT IS
CONSERVED IN A VARIETY OF
DIFFERENT PROTEINS INCLUDING
MSH 6 AND MSH 3, WHICH ARE HALF
OF THE COMPONENT ALONG WITH MSH2
OF EITHER MUTE S ALPHA OR MUTE S
DELTA AND ON THE RIGHT HAND SIDE
IS SHOWING THAT INVESTIGATING
THE ROLE OF PC NA AND MISMATCH
REPAIR AND COMPLICATED BY THE
FACT THAT AND I PULLED THIS OUT
OF THE LITERATURE, THIS IS THE
SEQUENCE ALIGNMENT OF THE PC NA
BINDING MOTIFS FOR HUMAN BINDING
PROTEINS AND THE LIST IS VERY
LONG.
DEFECTS IN BINDING, AND NOTED PC
NA, BECAUSE PC NA, BUT RATHER PC
NA BINDING MOTIF AND MSH3 AND
MSH6 AND ASK WHAT EFFECT IT HAS
ON REPLICATION FIDELITY.
SO WE'RE VIGOROUSLY INVOLVE
INDEED THAT PROCESS, AND WE
SHOULD HAVE SOME ANSWERS SOON TO
COMPLEMENT THE WORK IN MSH-6 AND
3 THAT'S BEEN DONE NOW FOR
SEVERAL YEARS, 1 OF THOSE IS
SECOND UNPUBLISHED OBSERVATION
THAT WE HAVE, THAT IF YOU TACK A
MUTATOR VARIANT OF POL DELTA,
THE L612 M-MUTANT AND YOU KNOCK
OUT MISMATCH REPAIR, BUT YOU
ONLY KNOCK OUT THE MS-3, H3
COMPONENT OF MUTE S BETA WHAT
YOU GET IS THERE'S A STRONG
MUTATOR PHENOTYPE THAT'S SORT OF
SELECTED FOR THE LOSS OF GC BASE
PAIRS IN HOMONUCLEOTIDE RUNS.
SO THIS IS 1 WAY OF SHOWING THAT
SO FAR WE BELIEVE THAT MSH3,
MEASURED BY MUTE O GENESIS AT
THE ERO 3 GENE IS A COMPONENT OF
MUTE S BETA THAT WHEN INACTIVE
LEADS TO MUTATIONS IN MINUS 1
BASE FRAME SHIFTS FROM GC RUNS
AT LEAST IN THE URO3 GENE AND
WE'RE CURRENTLY INVESTIGATING
THIS HYPOTHESIS USING A WHOLE
GENOME SEQUENCING TO SEE WHAT
THE SPECIFICITY ACROSS THE WHOLE
GENOME IS.
AND HERE IS AN EXAMPLE OF THAT.
IF YOU ENACTIVATE MUTE S ALPHA
BY KNOCKING OUT MSH-26, YOU GET
A MUTATOR PHENOTYPE THAT WAS
BASICALLY FOR ALL KINDS OF
ERRORS INCLUDING SUBSTITUTIONS,
1 BASE DELETIONS, 2 BASE
DELETIONS, 3 BASE DELETIONS, AND
IT'S SPECIFICITY IS UNUSUAL, IN
THAT AS THE DELETION SIZE GET
LARGER, THE RATIO OF ERRORS IN
MUTE S ALPHA VERSUS MUTE S BETA
CHANGES.
SO FOR SUBSTITUTIONS ONLY MUTSA
APPEARS TO WORK.
BUT FOR--ON THE LEFT-HAND SIDE
FOR LARGER DELETIONS, ONLY MUTSB
SEEMS TO WORK.
SO FOR EXAMPLE, FOR INSERTION
DELETION ERRORS ACROSS THE WHOLE
GENOME ARE GREATER THAN 1 BASE
PAIR, KNOCKING OUT MUTE S ALPHA
GIVES A 9 FOLD MUTATED PHENOTYPE
BUT KNOCKING OUT MUTE S BETA
GIVES 117 TOLD MUTATOR
PHENOTYPE.
SO WE'RE CURRENTLY CONTINUING TO
INVESTIGATE THIS TO CONFIRM OR
DENY THIS HYPOTHESIS, WHAT I CAN
TELL YOU IS NOW WE HAVE BETWEEN
50 AND 100,000 INDEL MUTATIONS
ARRIVING FROM THE WHOLE GENOME
SEQUENCING MACHINE WHICH WE CAN
LOOK AT THIS IN MUCH GREATER
DEPTH AND WE WILL BE PUBLISHING
ON THIS IN PROBABLY SOMETIME
2023.
THIS IS A SUMMARY OF WHAT I JUST
TOLD YOU.
I WILL LET YOU STARE AT THAT IF
YOU WOULD LIKE AND I WOULD BE
HAPPY TO ANSWER ANY QUESTIONS.
THANK YOU VERY MUCH.
>> THANK YOU VERY MUCH, TOM.
WE HAVE A COUPLE QUESTIONS HERE,
I WILL READ THE FIRST IF
DR. SOBEL, IS THERE ANY EVIDENCE
OF UNSYNTHESIZED DNA ORIGIN
FIRING SUCH AS THAT DUE TO ATR
INHIBITION AT ALL FOR FIDELITY
APPROVE READING CAPACITY.
>> TO MY KNOWLEDGE THE ANSWER IS
NO.
SO FAR OUR ABILITY TO ANSWER
THAT QUESTION COMES FROM WHOLE
GENOME SEQUENCING AND THERE WE
LOOK AT--WE CAN LOOK AT FOR
EXAMPLE, AN EARLY FIRING ORIGIN
AND SEE DIFFERENCES IN MUTE O
GENESIS FROM THE LATE FIRING
ORIGIN.
OR SEE DIFFERENCES IN
RIBONUCLEOTIDE CORPORATION BUT
WE DON'T HAVE THE RESOLUTION YET
TO LOOK AT INDIVIDUAL ORIGINS AT
THE KIND OF DEPTH THAT WOULD BE
NEEDED TO ANSWER THAT QUESTION.
>> THANK YOU.
STEVE LLOYD ASKS, THANKS FOR A
FANTASTIC OVERVIEW, MIX WIDE NEW
DATA, ARE THE FREQUENCIES OF
RIBONUCLEOTIDE INCORPORATION AND
REPAIR COMPARABLE BETWEEN
MITOCHONDRIAL DNA VARIOUSUS THAT
OBSERVED IN NUCLEAR DNA.
>> SO THAT'S A GREAT QUESTION, I
DIDN'T TALK ABOUT THE WORK IN
MITOCHONDRIA BECAUSE IT'S NOT MY
OWN AND ANDREW CLOSSON IN MY
LAB, LEFT THE LAB IN TWEBT 16, I
THINK IT WAS, MIGHT BE 2017 AND
JOINED UNIVERSITY OF
[INDISCERNIBLE] WHERE HE IS
CURRENTLY ASKING QUESTIONS ABOUT
RIBOYOU NUCLEOTIDE INCORPORATION
INTO THE MITOCHONDRIA GENOME IN
COLLABORATION WITH COLLEAGUES OF
HIS OWN AND WHEN WE LEFT HERE HE
AGREED THAT HE WOULD TAKE THAT
MITOCHONDRIAL FOCUS WITH HIM AND
THAT I WOULD NOT WORK ON IT.
BUT WHAT I WOULD SUGGEST IS
SINCE MITOCHONDRIAL RIBOYOU THIS
CLE O TIDE INCORPORATION OCCURS
VERY FREQUENTLY, BEEN SHOWN A
LONG TIME AGO, WITH THE
SPECIFICITY OF THAT KIND OF
RIBONUCLEOTIDE INCORPORATION
PRESUMABLY BY DNA POLYMERASE
GAMMA THE MITOCHONDRIAL REP
LIAISON CASE, IF YOU'RE
INTERESTED IN WHAT'S BECOME
FOUND OUT ABOUT THAT, I WOULD
HIGHLY RECOMMEND READING THE
ARTICLES THAT HAVE BEEN
PUBLISHED BY ANDERS CLOSSON OVER
THE LAST FEW YEARS, VERY
INTERESTING WORK BUT NOT OURS.
>> CAN I JUST FOLLOW UP ON THAT
QUESTION, AND ASK YOU, SO, IT'S
AMAZING HOW COMMON THESE RIPE O
NUCLEOTIDE INCORPORATIONS ARE
AND YOU SAY THAT THEY WILL LEAD
TO VARIOUS CONDITIONS AND
DISEASES AND CONCERNS, ARE THERE
ANY KNOWN ADDITIONS SORE
DISEASES FOR EXAMPLE, WHERE
THERE IS A PARTICULARLY HIGH
INCORPORATION OF RIBONUCLEOTIDES
ECISIONS WHERE THEY'RE
PARTICULARLY HIGH NUMBER OF
RIBONUCLEOTIDES THAT HAVE BEEN
INCORPORATED?
>> SO, I CAN GIVE YOU A VERY
GOOD EXAMPLE OF RIBONUCLEOTIDES
ARE INCORPORATE BIDE THE REP
LIAISON CASES, AND THEY'RE
NORMALLY REPAIRED BY
RIBONUCLEOTIDE EXCISION REPAIR
AND IF THAT KIND OF REPAIR IS
DISABLED, THAT RIBONUCLEOTIDE
EXCISION REPAIR AND THEY'RE LEFT
IN THE GENOME, THEY ARE SUBJECT
TO CLEAVAGE BY TOPOISOMERACE,
WHICH IS AN ENZYME THAT RELIEVES
SUPER COILS IN DNA AS PART OF
THE NORMAL REPLICATION PROCESS,
MOST OF THAT INCISION,
RIBONUCLEOTIDES BY RNA--WELL BY
TOPOISOMMER ACE 1 IS PRETTY
ACCURATE.
BHU IFLET RIBONUCLEOTIDE IS
REPETITIVE IN THE ELEMENT THEN
ISOMER ACE CAN CLEAVE THE DNA
AND RESEAL IT AFTER A SECOND
CLEAVAGE WHICH LEADS TO DELETION
OF 2 TO 5 NUCLEOTIDES IN
REPETITIVE DNA SEQUENCES.
AND THERE IS A PULL LITERATURE
ON THE CAUSES OF CANCER, THE
MUTATIONAL CAUSES OF CANCER SO
PEOPLE ARE DOING RESEQUENCING OF
TUMOR DNA PRACTICES LOTS OF
DIFFERENT KINDS OF CANCER AND
DEFINING THE KINDS OF MUTATIONS
THAT ARE LIKELY TO BE
RESPONSIBLE FOR INDIVIDUAL KINDS
OF CANCER AND THEY PRESENT ERROR
SIGNATURES, VARIOUS KINDS OF
BASE SUBSTITUTIONS OR DELETIONS
AND 1 OF THOSE SIGNATURES IS THE
FREQUENT PRODUCTION OF 2-5 PAIR
BASE DELETIONS AND THE
IMPLICATION IS THAT THEY MAY BE
CAUSED BY CLEAVAGE OR
RIBONUCLEOTIDES BY TOPOISOMMER
ACE 1 AS PART OF THE NORMAL
PROCESS OF DNA REPLICATION
REPAIR.
THERE ARE OTHER INDICATORS THAT
RIBONUCLEOTIDES MAY BE IMPORTANT
IN CERTAIN IMMUNOLOGICAL
DISEASES, I DON'T FOLLOW THAT
CLOSE LOW BUT THE ANSWER TO YOUR
QUESTION IS DEFINITELY A YES AS
SUGGESTED BY THE MUTATIONAL
SIGNATURES.
>> OKAY, HERE'S ANOTHER
QUESTION.
>> QUESTION FROM MICHAEL
[INDISCERNIBLE]: IN LIGHT OF
YOUR INTEREST IN EVOLUTION, WHY
DO YOU THINK THAT THERE ARE
SEPARATE LEADING AND LAGGING
POLYMERASES APPROXIMATE POLD
SEEMS TO BE ABLE TO DO BOTH
LEADINGINGS AND LAGGING, GREAT
TALK.
>> YEAH, SO IT DEPENDS ON THE
ORGANISM, FOR INSTANCE THE MAJOR
AND LEADING BACTERIA E.COLI IS 1
ENZYME DNA POLYMERASE, 3 SO IT
DOESN'T JUST FROM COMPARING THAT
TO WORK DONE IN EUKARYOTIC
NUCLEOTIDES CLER DNA
REPLICATION, THE CONSERVATION IS
NOT PERFECT.
WE--WHY THERE IS A LEADING
STRAND REP LIAISON CASE IN
UCARIOTS THAT DIFFERS FROM THE
LAGGING STRAND PROBABLY REFLECTS
THE NATURE OF THE REPLICATION
THAT OCCURS SO THE LEADING
STRAND, 60 SIS BY POLE IS
PRIMARILY THOUGHT TO BE LARGELY
CONTINUOUS WITHOUT INTERVENING
DISASSOCIATION POL E GRABS ON AT
ORIGIN AND SYNTH CISES THOUSANDS
OF NUCLEOTIDES WITHOUT EVERY SIS
ASSOCIATED UNDER NATURAL DNA
CONDITIONS WHEREAS THE LAGS
STRAND HAS TO BE REPLICATED BY
CONTINUOUS INITIATION BY POLA
FOLLOWED BY LESS HIGHLY
POSSESSIVE BUT NOT NEARLY AS
POSSESSIVE OF SYNTHESIS BY POL
DA WHICH HAS TO TO OKZAKI THAT
SEAL THOSE REGIONS OF DNA BUT
IN.
WHY THAT HAS TO BE DIFFERENT ON
AN AFLIEWGZARY TIME FRAME IS
CERTAINLY AN INTERESTING
QUESTION THAT WE ALREADY KNOW
EXCEPTIONS AND MIGHT NOT BE
COMPLETELY TRUE BUT IT IS
INTERESTING TO THINK HOW FEW
ORGANISMS WE ACTUALLY KNOW WHAT
THE MECHANISM OF DNA REPLICATION
SO THE--WE'RE USING YEAST AS A
EUKARYOTIC NUCLEAR REP LIAISON
CASE AND WE THINK THERE ARE A
LOT OF SIMILARITIES BETWEEN
YEAST AND HUMANS AND MICE AND
OTHERS EUKARYOTIC NUCLEAR
SYSTEMS BUT WHETHER THAT'SA
COMPLETELY CONSERVED
EVOLUTIONARILY IS SIMPLY NOT
KNOWN YET BUT IT'S OUR WORKING
HYPOTHESIS AS TO--MIKE ASKED A
GREAT QUESTION, WHY, WHY ARE
THEY DIFFERENT?
FOR THOSE THAT ARE DRNT, I DON'T
KNOW WHY.
>> THANK YOU.
AND THEN WE HAVE A QUESTION FROM
STEPHEN LLOYD, RELATIVE TO MAP
ORIGINS OF REPLICATION, ARE
THERE DISTRIBUTIONS EVER
POTENTIAL G-QUADROUGH ATOM PLEX
SEQUENCES RANDOM OR CLUSTERED?
ARE THESE DISTRIBUTIONS?
>> YEAH, SO ... THE ANSWER TO
THAT MUST BE NO BUT I'M NOT
ENOUGH OF AN EXPERT ON
G-QUADROUGH ATOM PLEXS TO KNOW
ABOUT HOW RANDOM OR NONRANDOM
THEIR DISTRIBUTION IS IN THE
GENOME.
THAT WOULD TAKE ASKING THAT
QUESTION OF THE EXPERTS ON THAT
SUBJECT.
I DO KNOW THEY ARE HIGHLY
SPECIALIZED SEQUENCES,
REPLICATION, SO WHEN WE DO WHOLE
GENOME SEQUENCING EVENTUALLY WE
ARE GOING TO LACK AT FIDELITY
ISSUES RELATED TO GQUADROUGH
ATOM PLEXS VERSUS NORMAL
NONQUADROUGH ATOM PLEX
CONTAINING DNA, BUT RIGHT NOW,
OUR WHOLE GENOME SEQUENCING DATA
NEED TO BE INCREASED IN REPORTER
DENSITY IN ORDER TO HAVE ENOUGH
MUTATIONS IN THE GENOME TO START
INVESTIGATING THOSE VERY
IMPORTANT QUESTIONS SO I
HONESTLY DON'T KNOW BUT THE
PEOPLE WHO STUDY G-QUADROUGH
ATOM PLEXES AND SHOW SOME OF IT
ARE IN THIS AUDIENCE WOULD BE
BETTER TO ASK THAN ME.
I HAVE A QUESTION OF MY OWN, YOU
ARE USING SEQUENCING TECHNIQUES,
WHOLE GENOME SEQUENCING WHICH
USES POLYMERASES ITSELF, TO
MEASURE AREAS IN POLYMERASES AND
ALGORITHMS HAVE BEEN DEVELOPED
WHO OVERLOOK THE ERRORS THAT
INTRODUCED BY THE POLYMERASES IN
THE SEQUENCES, HOW DO YOU DEAL
WITH THAT?
>> THE MAIN WAY WE DEAL WITH IT
IS WHAT WE'RE DOING IS LOOKING
AT MUSEUM TAIGDZ RATES OVER
MULTIPLE PASSAGES ON A PETRI
DISH AND THEN ASKING WHAT ARE
THOSE, WHAT ARE THE SAME--WHAT
ARE THE RATES FOR THE SAME KIND
OF ERRORS WHEN THAT STRAIN WAS
NOT PASSAGE THROUGH SAY 900
REPLICATION CYCLES SO WE ALWAYS
INTERPRET OUR DATA AS BEING THE
CONSEQUENCE OF THOSE REPLICATION
ERRORS BECAUSE WHAT WE SUBTRACT
OUT OF THAT IS THE BACKGROUND
MUTATIONS THAT WERE THERE AT THE
START AND THOSE BACKGROUND
MUTATIONS THAT WERE THERE BEFORE
THOSE 900 GENERATIONS ENCOMPASS
THINGS LIKE DNA SEQUENCING
ERRORS IN THE PROCEDURES ITSELF.
>> WE HAVE A QUESTION FROM SEAN
[INDISCERNIBLE] HERE, DEAR
THOMAS REALLY GREAT TALK, THIS
IS THE LIGASE 1 MEDIATED OKAY
LIGATION KOWP ILLEGALS OF
VARIATIONS.
>> COULD YOU REPEAT THAT.
>> YEAH, REALLY GREAT TALK,
LIGASE 1 MEDIATED OKAZAKI
LIGATION FOR THE PC R TO START
ANOTHER FRAGMENT SYNTHESIS ON
THE LAGS STRAND?
I DON'T KNOW WE'VE NEVER LOOKED,
THE OKAZAKI FRAGMENT INVOLVES
THE PIECE THAT WAS SYNTHESIZED
BE THE PRIME END OF THAT
PRACTICING MENTORSHIP SKILL,
BEING LIGATED TO THE 1 THAT'S
COMPLETED SO IN TERMS OF PCA, PC
NA BEING LIMITED FOR INITIATION
OF ADDITIONAL OKAZAKI FRAGMENTS
I DON'T THINK THE DATA WE HAVE
ADDRESS THAT QUESTION.
COULD VERY WELL BE BUT I DON'T
KNOW.
>> HERE'S ANOTHER QUESTION BY
DR. ROTH: HAVE YOU LOOKED AT
THE KIND OF MUTATIONS FOR THE
LIGASE 1 ON THE OTHER SITE
MUTANT IN THE CONTEXT OF A FEN1
NULL BACKGROUND?
>> WE ARE DOING THAT AS WE
SPEAK.
BUT THE WHOLE GENOME SEQUENCING
EXPERIMENTS WHICH WE THINK WOULD
BE THE FIRST WAY TO ADDRESS THAT
QUESTION.
THEY TAKE QUITE A WHILE SO YOU
HAVE TO SPEND SOME TIME DOING
THE STRAIN CONSTRUCTIONS, THEN
YOU HAVE TO PASSAGE THEM THROUGH
A LARGE ENOUGH GENERATION OF
REPLICATION CYCLES TO GET ENOUGH
MUTANTS TO HAVE SOMETHING TO
SAY, THEN YOU HAVE TO SEQUENCE
THEM, THEN YOU HAVE TO DO THE
BIOINFORMATIC ANALYSIS AND THEN
WE'LL KNOW.
FOR EXAMPLE, SOME OF THE
EXPERIMENTS THAT WE JUST
INITIATED FOR EXAMPLE, I
MENTIONED 1 OF THE NEW POST DOCS
HERE, MAHINA, SHE IS CURRENTLY
DOING A WHOLE GENOME SEQUENCING
EXPERIMENT THAT WILL TAKE
PROBABLY BETWEEN 6 MONTHS AND A
YEAR OF PASSAGING CELLS ON A PET
-PETRI DISH FOR WE SEQUENCE
THE WHOLE NUMBER OF MUTANTS AND
GET A REQUESTY TO THE QUESTION
SHE'S ASKING.
SO THAT WAS A GREAT QUESTION BUT
I DON'T KNOW THE ANSWER YET.
>> OKAY, AND 1 LAST QUESTION
HERE, FROM ANITA
[INDISCERNIBLE], CDC 9 LIGASE
FUNCTIONS ALL IN MITOCHONDRIA,
WHAT ARE THE EFFECTS OF THE
LIGASE DEFICIENCIES YOU STUDY ON
THE MAINTENANCE OF MTDNA IN
YEAST CELLS, I GUESS YOU ALREADY
SAID SOMETHING ABOUT T.
>> SO I HONESTLY DON'T KNOW
BECAUSE AS I MENTIONED BEFORE,
WE'RE LEAVING THE THE
MITOCHONDRIAL ANALYSIS WHO
DECIDED IT WOULD BE A POST DOC
OF HIS INFORMATION SO WE'VE NOT
ASKED THAT QUESTION
EXPERIMENTALLY PERHAPS EXPPERS
HAS.
THE ONLY THING WE'RE DOING ON
MITOCHONDRIA IS COLLABORATION
WITH BILL COPELAND DOWN THE
HALL.
HE'S 1 OF THE WORLD'S EXPERTS ON
MITOCHONDRIAL DNA REPLICATION
AND OUR WORK WITH BILL HAS TO DO
WITH THE ORIGIN OF MUTATIONS IN
MITOCHONDRIAL DNA.
NOT RIBONUCLEOTIDE
INCORPORATION.
SO I'M SORRY, IICISM DEE DON'T
HAVE THE ANSWER.
>> OKAY, WELL, THANK YOU SO MUCH
AGAIN.
I THINK WE HAVE TO STOP HERE.
AND THAT WAS A WONDERFUL LECTURE
TOM.
>> THANK YOU.
>> YES, THANK YOU TOM, IT WAS AN
EXCELLENT REVIEW, WE'RE HOPING
WE WILL BE ABLE TO POST A NUMBER
OF THESE SLIDES WITH THESE
SUMMARIES ON IT AND PEOPLE CAN
GO AND REVIEW THEM AT THEIR
LEISURE, YOU WERE PRESENTING AN
INCREDIBLE AMOUNT OF DATA THAT
YOU'VE ACCUMULATED OVER 40 YEARS
AND YOU'RE VERY HUMBLY INDICATE
THAT THAT'S ONLY A SMALL PORTION
OF WHAT WE SEE NOW, SO WE SEE
WHY YOU'RE STILL EXCITED ABOUT
THIS AND STILL WORKING VERY
DILLIENTLY IN THE LAB AND
PLANNING EXPERIMENTS THAT WILL
TAKE SEVERAL YEARS TO COMPLETE.
SO--THAT REMINDS ME TO REMIND
STUDENTS IN THE AUDIENCE, IF
ANYBODY'S LOOKING FOR A POST DOC
AND YOU'RE INTERESTED WHAT I
JUST TALKED ABOUT YOU'RE MORE
THAN WELCOME TO CONTACT ME.
>> OKAY.
THANK YOU.
>> YOU'RE WELCOME.
>> GREAT TO SEE YOU.
>> ABSOLUTELY THANK YOU GUYS.