Genome Integrity and Cancer Prevention: Molecular Mechanisms of DNA Repair


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Transcript:
>> IT'S MY PLEASURE TO WELCOME
TO YOU THIS YEAR'S G. BURROUGHS
MIDER LECTURE.
OUR LECTURER TODAY IS WEU YANG,
SENIOR INVESTIGATEORIES AT
NIDDK, HONORING THE MEMORY OF G
G. BURROUGHS MIDER.
HE WAS THE N.I.H. DIRECTOR OF
LABORATORIES AND CLINICS FROM
1960 TO 1968 UNDER THE
LEADERSHIP MUCH JAMES SHANNON,
LEGENDARY N.I.H. DIRECTOR.
THERE WAS AN EXPANSION IN THE
SIZE OF THE INTRAMURAL RESEARCH
PROGRAM AND IN THE 1960s WERE
IF FOUR NOBEL LAUREATES WORKING
AT THE N.I.H. AT THE SAME TIME.
IT WAS AN AMAZING PERIOD.
THE LECTURE RECOGNIZES A
SCIENTIST WHO SIMPLY NIC EXEMPLIFIES
EXCELLENCE.
THEY ARE VETTED BY THE BOARD OF
DIRECTORS AND MOVED FORWARD FOR
THE DIRECTOR TO CONSIDER.
THIS YEAR'S LECTURER IS WEI
YANG, DEPUTY CHIEF IN THE
LABORATORY OF MOLECULAR BIOLOGY
IN NIDDK.
SHE'S USED CREATIVE STRUCTURAL
APPROACHES TO REVOLUTIONIZE HER
UNDERSTANDING OF THE
INTERACTION OF DNA WITH
PROTEINS DURING REPLICATION,
REPAIR AND RECOMBINATION.
SHE RECEIVED A B I FRO B.A. FROM STONEY
BOOK, Ph.D. FROM COLUMBIA,
AND POST DOC WITH TOM STITES AT
ISLE.
SHE'S BEEN IAT -- AT YALE.
DR. YANG HAS APPRECIATED THE
NEED FOR DETAILED CRYSTAL
GRAPHIC STUDIES OF THE WAY IN
WHICH DNA AND ENZYMES INVOLVED
IN REPLICATION REPAIR AND
RECOMBINATION INTERACT.
THIS WORK IS S. HA HAS PROVIDED NEW INIT
INTO MutL AND MutL,
POLYMERASE BOUND TO SUBSTRAITS,
SPECIFIC RECOMBINATION COMPLEX
AND STRUCTURE OF RNH BOUND TO
RNA, CHARACTERIZED BY A
ELEGANTION OF EL AGAINST
CHEMISTRY TO GAIN UNDERSTANDING
OF BIOLOGICAL FUNCTION.
HER TALKS AS YOU'LL SEE AND
THOSE WHO HAVE HEARD HER TALKS
BEFORE WILL UNDERSTAND THIS,
ARE MODELS OF CLEAR
COMMUNICATION AND USE AS THEY
AESTHETICALLY BEAUTIFUL
STRUCTURES TO CLARIFY NEW
BIOLOGICAL CONCEPTS.
DR. YANG'S WORK WACK
RECOGNIZES -- WAS RECOGNIZED BY
THE HODGKIN AWARD.
THE TALK IS MOLECULAR
MECHANISMS OF DNA REPAIR.
WEI?
>> THANK YOU, MIKE.
THANK YOU FOR INTRODUCING ME.
I WAS VERY SURPRISED WHEN DR.
GOTSMAN TOLD ME THE NEWS I WAS
SELECTED TO GIVE THIS YEAR'S
MIDER LECTURE.
AS ONE OF MY PROFESSORS SAID
MANY YEARS AGO, THE BEST AWARD
WE SCIENTISTS WORK FOR IS PEER
RECOGNITION, AND I FEEL VERY
HONORED AND HUMBLED TO RECEIVE
SUCH DISTINCTION BY MY PEERS AT
NIH AND THE DIRECTORS.
AND WHEN I'M PREPARING THIS
LECTURE, I COULDN'T STOP ASKING
MYSELF WHAT I HAVE DONE TRULY
MATTERS TO MEDICAL RESEARCH,
AND A COUPLE DAYS AGO I WAS
ALERTED BY MIKE LISTON THAT MY
TALK ANNOUNCED ON THE NIH
WEBSITE AT 3:00 A.M., FEBRUARY
22.
[ LAUGHTER ]
IT DAWNED ON ME WHAT REALLY
IS THAT WE ARE WILLINGLING TO
TO WORK HARD TO MAKE A
DIFFERENCE.
I'M GOING TO TELL BUT SOMETHING
RELATED TO DNA REPLICATION,
ESSENTIAL FOR CELL
PROLIFERATION.
FOR EVERY CYCLE OF CELLS TO
DIVIDE BEFORE THAT DNA HAS TO
BE -- JOH GENOMIC DNA HAS TO BE
REPLICATED, USING CHECKPOINTS
BEFORE GOING TO THE REPLICATION
CELL DIVISION.
IS HAS TO COMPLETE THE
REPLICATION TO SURVIVE.
IT'S CRITICAL.
AN ASSOCIATE WITH DNA
REPLICATION, WE STUDY TWO
EVENTS, TRANSLESION ISN'T
SITUATION IS NECESSARY BECAUSE
ALL DNA IS NOT PERFECT.
EXPOSED TO SUN, IT CAN BE CROSS
LINKED IF WE EAT TOO MUCH
BARBECUE FOOD, WE MAY INGEST
CARCINOGENNENED AT THE DNA BASE
GETS DAMAGED.
IT BLOCKS NORMAL DNA
PRELIMINARIES.
ON THE OTHER SIDE OF THE COIN,
DNA DNA IS MADE BY KLAN
PRELIMINARIES, IT'S KNOWN FOR
LOW ERRORS.
IN A GENOME OF 3 BILLION BASE
PAIRS, EVEN ERRORS IN THE RATIO
OF ONE IN TEN MILLION BASE
COPIED, WEI GOING T WE ARE GOING TO HAVE
TENS TO HUNDREDS LEFT IN THE
DNA PRELIMINARIES, AND THOSE
ARE TAKEN CARE OF BY MISMATCH
REPAIR.
TO BYPASS THE DNA BLOCKING
REPLICATION BLOCKING LESIONS,
TO REPAIR ERRORS MADE BY DNA
PRELIMINARIES, ARE TWO SIDES OF
THE SAME COIN.
THE PURPOSE IS TO KEEP THE
GENOME AT MINIMAL MUTATION, AS
EVERYONE KNOWS CANCER RISES
BECAUSE OF MUTATIONS, THE
PATHWAYS REPLICATE ON MUTATION
RATE AND CANCER INCIDENCE.
I WANT TO START MY TALK TELLING
YOU WHAT WE DO IN THE LAB,
USING X-RAY CRYSTALLOGRAPHY,
STARTING FROM A GENE IDENTIFIED
THAT'S CAUSING HUMAN DISEASE,
AND IF WEI LUCK WE ARE LUCKY WE HAVE
WONDERFUL PEOPLE THAT
DISCOVERED NO PATHWAYS AND
PROTEINS BEFORE THE PUBLISH.
I HAVE TWO VERY GOOD COLLEAGUES
STOPPING ME ON MISMATCH REPAIR
AT NIH, ROGER WINGATE AND PEGGY
SEE.
THE ONES WE IDENTIFY, WE
GENERATE ENOUGH PROTEIN TO MAKE
CRYSTALS, THEY MAY BE
BEAUTIFUL, MAYBE NOT SO PRETTY,
BUT THE IMPORTANT THING IS THAT
WE CAN DEFLECT X-RAY AND RECORD
THOSE SPOTS ON FILM OR ON
VARIOUS RECORDING DEVICES
NOWADAYS, IT'S VERY ADVANCED.
AND THEN USING THAT INFORMATION
WE CAN GENERATE AND BUILD
STRUCTURE MODEL AND START TO
INTERPRET WHAT THE MODEL MEANS
AND WHAT THE STRUCTURE TELLS US
HOW THE MOLECULE WORKS.
WITH ADVANCED TECHNOLOGY,
GROWING CRYSTALS BACK BECOMES
EASIER.
WE HAVE RADIATION SOURCE IN
CHICAGO, WE DON'T EVEN NEED TO
GO THERE.
TONIGHT WEI GOIN WE ARE GOING TO COLLECT
DATA IN THE OFFICE AND SHIP THE
CRYSTAL BY FED EX AND SPEND
MORE TIME TO UNDERSTAND
STRUCTURE.
IT'S PRETTY BUT DOESN'T TELL
US.
WHEN YOU SEE THE STRUCTURE, YOU
CAN'T UNDERSTAND HOW IT WORKS.
WE SPEND A LOT OF TIME TRYING
TO UNDERSTAND THAT, USING
BIOCHEMICAL CELL-BASED TOOLS,
AND THE OTHER THING, ADVANCED
TECHNOLOGY ALLOWS US TO INSTEAD
OF STUDYING ONE SINGLE SMALL
PROTEIN, NOW WE CAN STUDY LARGE
PROTEIN COMPLEXES, MADE OF MORE
PROTEINS BECAUSE NO PROTEIN
WORKS ALONE.
SO THE FIRST PART I'M GOING TO
FOCUS ON IT MISMATCH REPAIR,
REPAIRS THE MISMATCHING REPAIRS
BY DNA PRELIMINARIES.
HERE IS STORY FROM A FAMILY.
IT'S INHERITABLE.
ONE IN 20 COLON CANCER PATIENTS
DIAGNOSED IN AMERICA CARRY
MUTATIONS DEFICIENT IN IN
MISMATCH REPAIR.
ACCORDING TO THE AMERICAN
CANCER SOCIETY A MILLION OF
AMERICANS CARRY ONE OF THOSE
DEFECTIVE MISMATCH REPAIR GENES
AND THOSE PEOPLE HAVE 80% OF A
CHANCE TO DEVELOP CANCER IN
THEIR LIFETIME.
IT AFFECTS MANY OF US.
THE TWO GENES OR FOUR ENCODING
TWO SPECIAL PROTEINS CALLED
MutL AND MutS.
IT'S EASIER TO HANDLE, MutL
AND MutS ARE PRESERVED FROM
BACTERIA TO HUMAN.
HERE IS A DEMONSTRATION OF
BLACK SLIME, THE RE LINE, THE RED LINE.
THE MutS RECOGNIZES THE
SINGLE MAS MISMATCH AND RECOGNIZES
MutL.
THE NUCLEUS WILL MAKE AN
INCISION IN UNMETHYLATED
STRANDS AT THE SITE.
THE ONCONCE MADE, IT UNWINDS THE DNA,
BECAUSE THE SEQUENCE CAN BE IN
THE GENOME AND USUALLY 100 BASE
PAIR AWAY FROM THE MIS MISMATCH.
THE WHOLE STRAND WILL BE
RESYNTHESIZED IN THAT PATCH OF
100.
AS YOU CAN SEE, THE PROCESS IS
EXTREMELY EXPENSIVE.
THIS PROCESS IS CONSERVED ON
HUMANS AS WELL AS E. COLI AND
MutL IS PLAYING THE INTENTION
SAME ROLE, IN ADDITION THE
PROCESS ACTUALLY CONTAINS THREE
ATPA'S.
IATPA'S.
INTERESTINGLY, MutS IS KNOWN
FOR ABC TRANSPORTER, MutL IS
A GHL, A DIFFERENCE ATPA, AND
U.V. IS A BONE A SID BONA FIDE PROTEIN.
PEOPLE SAY ATP CAN DO MANY
THINGS, BUT YOU ASK MOST PEOPLE
WHAT YOU THINK ABOUT THAT,
PEOPLE SAY IT'S A MOTOR
PROTEIN.
WHAT I'M GOING TO SHOW YOU IS
MutL AND MutS ARE DOING
SOMETHING DIFFERENT.
WE CALL IT A SINKER, MutL WE
THINK IS A TALKER, RECRUITING
PROTEIN TO COME TO DO THE
REPAIR.
ONLY THE UVRD IS A TRUE MOTOR
PROTEIN.
I'LL START FROM THE SIMPLEST,
THAT'S THE FUNCTION EVERYONE
UNDERSTANDS.
MOTOR PROTEIN IS BURNING ATP,
THE ENERGY IS USED TO ORGANIZE
THE MOTION TO DIRECTIONAL
WALKING.
IT'S NOT ENOUGH TO MAKE
DIRECTIONAL MOVEMENT.
IF YOU DROP A PERSON INTO A
VIRGIN FOREST WITHOUT ANY PAST,
THE PERSON CAN WALK IN THE
FOREST FOR DAYS, NEVER GET OUT,
BECAUSE IT DOESN'T KNOW WHERE
GPS.LK TO UNLESS HE HAS A
IF THERE'S A PATH ONE CAN WALK
ON THE PATH AND GET OUT OF THE
FOREST.
AND THE MOTOR PROTEINS ARE LIKE
HUMANS.
IN ORDER FOR THEM TO WALK IN
CERTAIN DIRECTIONS THEY NEED A
TRACK, SO EVERYONE KNOWS THE
FILAMENT, ACTING FILAMENT IS
THE TRACK.
CRYSTALLOGRAPHY.O LARGE FOR
HERE IS A PICTURE USING ATM AND
VIDEO RECORDING IN JAPAN.
THAT'S THE STATE OF ART RIGHT
NOW, YOU CAN WATCH A MOTOR
MOVE.
YOU CAN SEE IT'S HARD TO REALLY
SEE THE DETAIL.
ONE WALKS QUICKLY, ONE GETS
STUCK.
YOU DON'T KNOW WHAT HAPPENS
THERE.
IT'S JUST LIKE USING A WATCH,
IF THE WATCH IS TELLING YOU THE
PERFECT TIME, YOU'RE SATISFIED.
BUT IF THE WATCH IS TELLING YOU
WRONG TIME, OR DOESN'T WORK,
YOU NEED TO LOOK A LITTLE
CLOSER.
OPEN THE WATCH, LOOK AT THE
DETAIL.
TO REALLY UNDERSTAND HOW THE
PROTEIN WORKS WE NEED A CLOSER
LOOK.
HOWEVER, THE TIME RESOLUTION
AND SPATIAL RESOLUTION REALLY
IS GOING AGAINST ONE ANOTHER.
IF YOU WANT TO SEE THE REAL
TIME, YOU HAVE TO DROP THE
RESOLUTION.
IF YOU WANT TO SEE THE HIGH
RESOLUTION LIKE X-RAY
CRYSTALLOGRAPHY WE NEVER CAN
RECORD A DYNAMIC PROCESS
BECAUSE WE PROCESS ONE AT A
TIME.
THAT INCOMPATIBILITY IT BE
RESOLVED, USING THE EXAMPLE BY
EDWARD MAYBRIDGE, PROBABLY MANY
PEOPLE HAVE SEEN THIS MOVIE, IT
LOOKS BEAUTIFUL.
IT WAS RECORDED.
WAY BEFORE THERE WAS VIDEO
CAMERA.
IT WAS DONE IN 1878.
AND THE WAY HE MADE THIS MOVIE,
THE FRAMES PER SECOND ARE
FEWER.
HE MADE IT BY PLACING CAMERAS
AROUND THE RACETRACK AND THOSE
PICTURES, HE MAY HAVE SORTED
THEM AND PUT THEM TOGETHER TO
MAKE A STOP ACTION MOVIE BUT
THAT GIVES THE IDEA
CRYSTALLOGRAPHY CAN USE THE
SAME WAY TO MAKE MOVIES.
WE MAY NOT BE ABLE TO MAKE A
PANEL OF THE PICTURES BUT MAYBE
TWO OR THREE VERY ESSENTIAL
STAGES OF THE MOLECULE, HOW
THAT WORKS.
AND THEN SPLICE THEM TOGETHER
TO MAKE A MOVIE.
THAT'S EXACTLY WHAT WE DID.
SO WE CRYSTALLIZED UVRD WITH
DNA AND CRYSTALLIZED WITH DNA
AND WITH A NONHYDROLZABLE.
HYDROLYSIS., A HIGH DROLL SIS
IF YOU COULDN'T THE BASE PAR
FROM THIS POINT TO THAT POINT,
THEY ARE DIFFERENT.
BINDING ADP AND NOT BINDING
ADP.
I WILL STRING IT TOGETHER AS A
MOVIE.
THE WORK WAS DONE REALLY
THOROUGHLY BY A POST DOC, J
ALLEN LEE, WHO IDENTIFIED THE
THREE STATES.
NOW YOU CAN WATCH.
ATP COMES IN AND INDUCES
EMOTION BETWEEN THIS PORTION OF
THE PROTEIN TO THAT PORTION OF
THE PROTEIN.
IT'S A ROTATION.
DNA IN THE SCREW MOTION COMES
DOWN AND PUSHES AGAINST THIS,
HAIRHE BASE CLOSER TO THE PAIR
PIN GETS SEPARATED.
THE ADPI RELEASE MOTION.
WE BREAK DOWN THE ATPA CYCLE,
BINDING INDUCED THE UNSCREWING
OF THE DOUBLE STRAND AND THE
ONE BASE GETS IMPAIRED.
AND AFTER ATP HIG HYDROLYSIS, IT
ALLOWS SINGLE STRAND LOCATION
TO COMPLETE THE CYCLE BY MAKING
THE THREE-STATE STRUCTURE
OBSERVATION AND PUT THEM
TOGETHER, WE CAN ANSWER WITH
CONFIDENCE, AND WE KNOW WHERE
THE PROTEIN DNA OCCURS.
SOME DAY WE'LL BE ABLE TO USE
IT TO REALLY HELP THE HUMAN
UNDERSTAND HOW IT WORKS IN THE
HUMAN BODY.
THE WORK WE DID LEER IS U HERE IS UP TO
DATE.
PROBABLY IT'S STILL THE BEST
STRUCTURE DESCRIPTION OF HOW IT
WORKS.
NOW I WILL SWITCH GEARS TO THE
SECOND ATP, MutL, IT'S A
DIMER.
350 RESIDUE, VERY CONSERVED.
WE SAID LET'S CRYSTALLIZE IT
AND SEE HOW IT WORKS.
THE POST DOC, MY FIRST POST DOC
DID THE WORK.
HE SOLVED THE STRUCTURE.
ACTUALLY AFTER THE STRUCTURE
WAS SOLVED WE WERE QUITE
DISAPPOINTED.
THE REASON IS THAT OUT OF 350
RESIDUES THAT'S CRYSTALLIZED WE
ONLY CAN SEE 280.
70 WERE DISORDERED.
MANY ARE CONSERVED.
SO WE HAD TO LEAVE THE
STRUCTURE ON THE SHELF FOR A
WHILE, TRUE TO UNDERSTAND WHAT
THE STRUCTURE MEANS.
WHILE WE WERE BUSY DOING
BIOCHEMISTRY WE STUMBLED ON
THAT, THIS PROTEIN IS AN ADPA,
IN THE LIT A FUEL I LITERATURE IT WAS
LABELED WITH NO ENZYM ENZYMEATIC
ACTIVITY.
THERE WERE TWO POWERS, ONE FROM
NIH, IDENTIFIED THE MutL
SEQUENCE HEMOLOGY.
THE DNA GYRASE WAS DISCOVERED
ON THIS CAMPUS BY MY
COLLEAGUES.
SO WE WENT AHEAD AND
RECRYSTALLIZED A PROTEIN IN THE
PRESENCE OF ATP.
NOW THE STRUCTURE LOOKS
DIFFERENT FROM WITHOUT ATP.
THE ONE SHOWING ORANGE, THE 50,
60 RESIDUES NOW UPON ATP
BINDING BECOME ORDERED.
SOME OF THOSE SECOND STRUCTURE
FORMED INTERFACE FOR THE
PROTEIN TO DIMERIZE.
THIS SCHEME OF CHANGE ACTUALLY
IS APPLICABLE TO THE WHOLE
FAMILY OF THIS AND T-SHOCK
PROTEIN 90.
SO I'M JUST RECAPITULATING THIS
MOVEMENT OF ATP INDUCED CHANGE
IN THE MOVIE SHOWING YOU AGAIN
THE PROTEIN STARTING LOOKING
PATHETIC, IT'S MISSING A LOT OF
THINGS.
ONCE ATP BONDS, YOU SEE INDUCED
A HUGE AMOUNT OF CONFIRMATIONAL
CHANGE, SECONDARY STRUCTURE
FORMATION, THE PROTEIN GOES
FROM MONIMER TO DIMER.
IT WAS DIFFICULT TO IDENTIFY
THIS IS AN ATPA, PROTEIN 90 WAS
NOT KNOWN, DENIED AS AN ATPA
FOR YEARS.
THE REASON THE ATP TURNOVER
RATE IS SLOW, MUTILE TURNOVER
ATP ONCE PER FIVE MINUTES.
WHAT IS IT DOING IN FIVE
MINUTES?
IT TURNS OVER SO FULLY.
WE WERE LOOKING FOR WHAT CAN
CONTROL THE SPEED OF ATP
HYDROLYSIS.
WE MADE A BUNCH OF MUTATIONS
ALONG THE PATHWAY, BLOCKED FROM
ATP BINDING, BLOCKED
DIMERIZATION, ATP HYDROLYSIS.
WHEN THE MUTILES BIND IT'S THE
ONLY STATE IT CAN ACTIVATE.
THE DNA HAS SINGLE STRAND
EXPOSED, SPEEDING UP THIS
PORTION AND REDUCED TIME HERE,
INCREASED TIME IN THE FREE
FORM.
AND THE WHOLE PROTEIN IN THE
WHOLE CYCLE OF ATH HYDROLYSIS
IS ESSENTIAL FOR ACTIVATING.
IT NEEDS A SIGNAL AND NEEDS TO
BE HEALTH TO DO IT IN HUNDRED
BASED PAIRS.
HERE IS A MODEL WE THINK HOW IT
WORKS.
IT'S TURNING OVER QUICKLY AND
UNWINDING HUNDREDS PER MINUTE.
THE MUTILES HOLDING IT TOGETHER
ARE DNA, AND ITSELF TURNING
OVER ONCE IN A FEW MINUTES.
IT'S ONLY WHEN THE DNA BECOMES
TANGLED BECAUS, REACTIVATED IF NEEDED
AGAIN.
SO THOSE TWO STORIES ARE
RELATIVELY STILL STRAIGHT
FORWARD, CHANGING STRUCTURE,
SERVICING AN ENERGY SOURCE TO
ALLOW A MOTOR TO WORK.
I'M GOING TO THE MOST
INTRIGUING ATPA OF THE FAMILY,
THE ABC FAMILY.
IT'S RECOGNIZING DNA MISMATCH.
THERE ARE TWO TYPES OF AREAS
THAT DNA PRELIMINARIES MAKE.
ONE IS MISSING THE BASE AGAINST
THE TEMPLATE.
MICROANS WE HAVE MIKE ROY
SATELLITE, IT MAY SKIP FORWARD
OR BACKWARD RESULTING IN LOOPS
IN DNA.
THOSTHE MISS MATCH INWE NEED TO FIND THEM .
THE RATIO OF OCCURRENCE,
FREQUENCE OF OCCURRENCE OF THE
MISMATCH IS ABOUT ONE IN TEN
MILLION BASE, YOU THINK ABOUT
IT.
THE PROTEINS SEARCH THROUGH THE
GENOME, ONCE FINDING IT, ONE IN
TEN MILLION BASE PAIRS, TO FIND
THE MISMATCH, WHICH LOOK VERY
DIFFERENT ON NORMAL PAIRS, IT'S
NOT GC BASED, IT'S A TREMENDOUS
TASK, HOW TO FIND THIS ONE
MISMATCH BURIED IN TENS OF
MILLIONS OF NORMAL BASE PAIRS.
IT'S VERY REASONABLE TO LIKEN
IT TO FINDING A NEEDLE IN A
HAYSTACK.
THAT'S INTRIGUING, THE MISMATCH
HAS DIFFERENT SHAPES.
I WAS APPROACHED, WONDERFUL
PROTEIN, HEAT RESISTANT, HER
POST DOC EVEN BETTER.
WHILE WE WERE SIDETRACKED BY
SOME CRYSTAL THAT'S NOT
DEFLECTING WELL, GALIA SLOWED A
CRYSTAL AND WE SOLVED THE
STRUCTURE.
WE WERE THE FIRST ONE THAT
SOLVED EVER THE TEST OF THE DNA
TOGETHER, IT'S A FAIRLY LARGE
PROTEIN, TWO SUBUNITS, GREEN,
BLUE.
BLUE IS THE ONE SHOWING
INTERACTING WITH THE MISMATCH,
BECAUSE EVEN THOUGH IT'S A
HOMO-DIMER, THE STRUCTURE ARE
DIFFERENT BECAUSE THERE'S ONLY
ONE DNA STRAND THAT CONTAINS
THE MISMATCH.
THE BLUE SUBUNIT IS INTERACTING
WITH THE MISMATCH HERE, BUT
EACH SUBUNIT HAS TWO DOMAINS,
ONE ON TOP, ONE BELOW HOLDING
THE DNA TOGETHER.
THE DNA IS QUITE BENT.
AND WE DETERMINED THE DIFFERENT
MISMATCH, WE PUBLISHED TOGETHER
BINDINGb
MISMATCH.
IT LOOK JUST THE SAME.
SEVEN YEARS LATER, HUMAN
PROTEIN THAT CAUSED COLON
CANCER, IT LOOKED SIMILAR.
IN HUMANS BLUE AND GREEN ARE
SEQUENCE RELATED BUT NOT
EXACTLY THE SAME, THE BLUE
SUBUNIT AGAIN RECOGNIZED THE
MISMATCH.
AND AFTER SOLVING THE
STRUCTURE, OF COURSE, I WILL
TELL YOU THE MOLECULAR MEMBER
MECHANISM.
DNA IS BENT.
THE SECOND THING IS THAT HOW
DOES IT INCREASE THE
SPECIFICITY, BECAUSE THE
DIFFERENT TO BIND IN NORMAL
DNA, IT'S ONLY HUNDRED-FOLD,
NOT ENOUGH TO EXPLAIN FINDING A
MISMATCH IN 10,000 OF THE
NORMAL BASE PAIRS.
TURNS OUT IT'S AN ABC ATP
FAMILY.
YOU WOULD THINK MAYBE ADDING
ATP THEN THE PROTEIN WOULD BIND
TO THE MISMATCH MORE TIGHTLY
BUT EXACTLY THE OPPOSITE.
THERE WAS ALL KINDS OF
PROPOSALS THAT MutS IS THE
MODE OF PROTEIN, IT MOVES AWAY
TO FINDING NEW CLEAVAGE SIDES
OR DOESN'T HYDROLIZE.
THE SEAMLESS STRUCTURE THAT DNA
IS BENT, WE DON'T THINK IT CAN
SLIDE SO EASILY, ALSO IF IT
LEAVES MISMATCHED WHAT WILL
HAPPEN TO THE MISMATCH IS THERE
A SECOND COMING, A HUGE MESS
THERE?
AND WHY SHOULDN'T IT STAY?
SO THE POST DOC DESIGNED THIS
EXPERIMENT TO TEST WHETHER
MutS NEEDS MISMATCH TO
ACTIVATE THE REPAIR PROCESS.
NORMALLY THE DNA ARE CONNECTED.
THERE'S A MISMATCH AT THE
CLEAVAGE SITE, IF THE PROTEIN
HAS TO TRAVEL ALONG DNA
HYDROLYZING TO COME TO THE SIDE
TO ACTIVATE NUCLEASE, ENABLED
ACTIVATING MISMATCH REPAIR.
AND AS A RESULT, AFTE IT REQUIRES
BRIDGE IT.GIT, SUGGESTING
LATER ON JOHN DID A DIFFERENT
EXPERIMENT USING HUMAN PROTEIN.
HE BLOCKED THE DNA PATH USING
EVIDENCE AND SHOWED AGAIN THAT
MutS CAN ACTIVATE IN THE
PRESENCE OF A BLOCKER.
THE KINETICS OF ACTIVATION IS
THE SAME.
FROM THE HIGH ENERGY CO-FACTOR
USED IN PROTEIN SYNTHESIS,
USING GTP TO INCREASE THE
SPECIFICITY OF AMINO SELECTION.
SO THE IDEA ACTUALLY WAS --
CAME OUT WHEN I WAS TALKING
ABOUT THIS PROBLEM.
SO WE PROPOSED THIS MODEL THAT
MutS BINDS TO NORMAL PROTEIN
AND ATP HELPS IT RELEASE SO IT
HAS A CHANCE TO GET THE
MISMATCH.
ONCE THERE, IT CAN RECRUIT
MutL.
THE TWO TOGETHER NOW IN THE
PRESENCE OF ATP CAN BIND DNA
MISMATCH.
THIS DATA IS GENERATED BY MANY
LABS.
THE MODEL WE PROPOSED IS STILL
IN THE TESTING STAGE, AND WE
STILL CANNOT EXACTLY TELL WHY
IT BANDED TO MISMATCHED AND IS
DIFFERENT.
WHEN THE TWO TOGETHER BEING
SHOWN BY MANY GROUPS CAN
ACTIVATE IN THE NUK NUCLEUS AND BY
US IN EITHER CASE.
IT WAS A POSITIVEEL FOR MORE
THAN TEN YEARS.ZZLE FOR MORE
A POST DOC IN MARTY'S LAB AND
WORKED WITH ME AS WELL SOLVED
THE STRUCTURE OF HUMAN MutS
BETA, RECOGNIZING DNA THAT'S
NOT A SIMPLE MISSED PAIR BUT
HAS LARGE BASED LOOPS THAT HAVE
NO PARTNER.
AND SHE WORK THE DILIGENTLY FOR
FOUR YEARS AND SOLVED THE
MutS COMPLEX WITH DNA
CONTAINED TWO, THREE, FOUR, AND
SIX, FOUR CRYSTAL STRUCTURES,
THE REASON WE SOLVED MANY
STRUCTURES, SOMETIMES CRYSTAL
STRUCTURES ARE NOT AS UNBIASED,
BECAUSE OF INTERACTION, HAVING
POOR STRUCTURE WE HAVE A
COMPLETE VIEW.
HERE IS THE ORIGINAL STRUCTURE
OF BACTERIA IN HUMANS, MutS
RECOGNIZES MISSED PAIR OR
SINGLE IMPAIRED BASE.
THIS. THE STRUCTURE LOOKS LIKE7]
THE FIRST VIEW DOESN'T LOOK
THAT DIFFERENT EXCEPT AT THE
TOP LIKE A CORKSCREW, NOW IT'S
TIGHTER, TWINED BETWEEN THE
GREEN AND BLUE SUBUNIT.
THIS PORTION IS IS CONSERVING
ALL MutS PROTEIN EXCEPT WHEN
WE WERE STUDYING BACTERIA
PROTEIN WE REMOVED IT FOR EASY
CRYSTALLIZATION, AND IN THE
MutS STRUCTURE THIS GETS
DISORDERED.
THE REAL DIFFERENCE, I'LL SHOW
YOU IN THE OTHER SLIDE WHEN WE
ZOOM IN, HERE IT DOESN'T REALLY
SHOW.
SO THERE ARE A DIFFERENT NUMBER
OF BASE IMPAIRED IN THE LOOP.
WEI SHOWING YOU TWO AND FOUR
AND SIX.
WHAT YOU SEE IS WHEN THE LOOP
SIZE CHANGES, THE DNA ANGLE IS
DIFFERENT.
AND THE ANGLE IS PROPORTION TO
THE LOOP SIZE.
ALL THE DIFFERENT SIZES OF LOOP
ARE RECOGNIZED IN THE REPAIRS.
EXACTLY THE DNA SHAPE MAY NOT
BE IMPORTANT, HOW MUCH IT'S
BENT IS NOT IMPORTANT.
THE SECOND THING WE LEARNED
FROM HERE IS THAT MutS EVEN
GIVES TWO BASE PAIR IMPAIRED,
SAME DNA SUBSTRATE, TO IMPAIR
THE BASE ALSO FLIPPED OUT IN
OPPOSITE DIRECTIONS.
SO EXACTLY WHERE THE MISMATCH
LOCATES RELATIVE TO THE PROTEIN
IS NOT EVEN IMPORTANT, BECAUSE
THEY ARE REPAIRED BY DIFFERENT
MutS.
WHAT DOES IT RECOGNIZE?
CLEARLY ONE COMMON FEATURE YOU
SEE IS THAT THE DNA IS BENT.
IT'S BENT BY DIFFERENT ANGLES.
THAT COMES TO AN ANALOGY, MAYBE
A GOOD DNA IS JUST A GOOD
SPRING PERFECT.
THE LESION WHICH IS TRUE FOR
ANY DNA LESION, WHEN THERE'S A
LESION, THE SPRING IS NO LONGER
AS RIGID.
IT WELCOMES MORE BENDABLE.
HOW DO YOU SPELL THE SPRING IS
PERFECT, THAT YOU CAN STRETCH
OR GO BACK, IF YOU STRETCH AND
BROKE IT IT WON'T BE BACK.
THE BEST WAY IS JUMPING OUT,
GIVE IT ENERGY TO SEE IF IT CAN
RESIST AND BOUNCE BACK.
IF SO, THAT'S WHAT WE THINK
MutS IS DOING.
WE KNOW WHAT TO LOOK FOR, WE
FIND THE DIFFERENCE.
THERE ARE TWO SUBUNITS.
ON THE RIGHT IT'S MULTI-COLOR
BUT REMEMBERS THE BLUE
SUB-UNIT, DNA IS CONTAINING A
MISMATCH.
ON THE LEFT, IT'S THE GREEN
SUB-UNIT.
THE DIFFERENCE IS IN THE DNA
BINDING DOMAIN.
YOU SEE THAT THE ONE, THIS
DOMAIN, BLUE, IS WAY DOWN.
THIS SUBUNIT IS NOT INVOLVING
DIRECT READING OF MISMATCH.
IT'S WAY UP.
IT'S LIKE A CRANE.
STRAIGHT STANDING UP, THE OTHER
LEG IS BENT.
THE STANDING ONE IS LOOKING FOR
BENT, THEDNA CAN BE WEPT AND
OTHER IS FLEXIBLE.
WHAT'S REALLY INTERESTING IS
THIS SUB-UNIT IN PRESENT IN
BOTH HUMANS MutS, ALPHA AND
BETA.
THEY HAVE DIFFERENT
CONFIRMATIONS.
IT'S FREE TO MOVE WITHOUT
DIRECTLY INTERACTING WITH THE
MISMATCH.
ABLE TO MOVE LIKE THIS, THAT
THIS AFFECTS HYDROLYZED ATP.
THIS SUBUNIT IS PULLED DOWN,
THE ABC FAMILY IS OCCLUDED FOR
HTP BINDING LEADS TO THE
EXPLANATION WHY THE KINETICS IS
DIFFERENT.
WE WERE ONE OF THE GROUPS, WE
NEVER PUBLISHED IT BECAUSE WE
WERE BEHIND, BUT IT'S ALWAYS A
PUZZLE THAT MutS WILL
HYDROLYZE ATP WELL.
WITH NORMAL DNA, HYDROLYZE
THIS.
IF YOU PRESENT A MISMATCHED
DNA, THE BIRTH PLAC FIRST PHASE IS
ELIMINATED, ANY STEP BEFORE
HYDROOLYSIS IS SLOW.
WE HAVE AN ANSWER.
IT'S BINDING TO THE NORMAL DNA,
THE DNA IS LIKE A GOOD SCREEN,
RESISTS BEING BENT.
THIS DOMAIN IS TRYING TO BOUNCE
ON IT, AND THE DNA WOULDN'T
BUDGE.
IT'S SLIDING ALONG, THE TWO ATP
HYDROLYSIS GOES ON STRONGLY.
ONCE IT BINDS TO A MISMATCH, IT
GETS PULLED DOWN AND IT CANNOT
BIND ATP WELL.
THE GREEN SUB-UNIT CAN BOUNCE
IS SLOWER.ROLYSIS IS JOE
WE HAVE A MODEL HOW MutS
IDENTIFIES MISMATCH, BECAUSE
USING THE ATP AS A PROOF
READER, IT BINDS NORMAL DNA,
TRIES TO BEND IT, AND DNA IS
VERY STRONG, RESISTS TO
BENDING, ALLOWS ATP HYDRO LYSIS
TO GO ON STRONGLY, SLIDING
ALONG THE DNA.
AND IF IT REACHES THE MISMATCH,
BY DIRECT ASSOCIATION OR BY
SLIDING, BECAUSE THE MISMATCH,
THE DNA CAN BE INTENT, THE BLUE
FINDS EASY FOOTING.
ONCE IT GETS STUCK ON THIS, THE
ATP IS INHIBITED, THE
HYDROLYSIS IS NOT ENOUGH TO
DISASSOCIATE IT, AT LEAST FOR A
TIME DURATION, AND BY GREEN
SUB-UNIT CAP CAPABLE OF HYDROLYZING
THE TWO RECRUIT THE NEXT
PROTEIN FORMING THE PAIR
COMPLEX.
WE ARE DOING EXPERIMENTS, A LOT
OF PEOPLE ARE OUT IN THE WORLD
DOING EXPERIMENTS, EITHER TO
PROVE OR DISPROVE IT, BUT SO
FAR IT'S SATISFYING GREEN WITH
KINETIC DATA AND STRUCTURAL
DATA.
NOW I'M GOING TO RETURN TO THE
POINT WHEN WE STARTED, THE
CANCER.
HOW IT RELATED COLON CANCER AND
HOW IT'S HELPING KNOWING THE
MOLECULAR MECHANISMS TO HELP
UNDERSTAND THE CANCER
MECHANISM.
SO IT WAS REALLY NICE TO HAVE
THE WHOLE POOL OF DATA,
HUNDREDS OF THOUSANDS OF DATA
POINTS, MUTATIONS FOUND IN THE
CANCER PATIENT.
SO ONCE WE HAVE STRUCTURE WE
CAN PROJECT THEM WHERE THEY
ARE.
AND WHEREVER THERE'S COLON
CANCER, THAT'S FUNCTIONALLY
IMPORTANT.
IT WASN'T THAT WE ALWAYS CAN GO
TO THE RIGHT PLACE.
WE FIRST FOUND IT, IMMEDIATELY
IT'S NOTICED THE ATP SITE IS
IMPORTANT.
BECAUSE A LOT OF MUTATIONS
CAUSE CANCER, AND THEN THIS IS
A HELIX LINK TO DNA BINDING,
ALSO CAUGHT OUR ATTENTION.
ONLY RECENTLY WE NOTICED THIS
BOUNCING EFFECT OF THE DNA
BINDING.
WE APPRECIATE THIS INTERDOMAIN
CONNECTION INTERFACE ARE SO
IMPORTANT FOR COMMUNICATING THE
ATP ACTIVITY TO DNA BINDING,
AND ALLOW THE ATP ENERGY USED
TO TEST WHETHER THE DNA IS
CONTAINING A MISMATCH OR NOT.
AND FURTHERMORE, WE KNOW THAT
NOW, THAT BETWEEN THE
COMMUNICATION BETWEEN TWO
SUBUNITS, THE ENTE INTERFACE IS
IMPORTANT.
WE CAN ONLY PROPOSE MAYBE IT'S
IMPORTANT FOR BINDING TO THE
MutL OR OTHER FACTORS IN
MISMATCH REPAIR.
THAT'S HOW WE USE THE DATA, IN
TERMS OF HELPING, IT'S NOT A
GOOD DRUG TARGET BECAUSE IT'S
MALFUNCTIONING, THAT'S WHY WE
HAVE CANCER.
TO THE STRUCTURE OF THE
MOLECULAR MECHANISM, NOW IF WE
FIND A MUTATION, WE CAN HELP TO
IDENTIFY WHETHER THE MUTATION
ACTUALLY IS TRULY CAUSING
CANCER OR MAYBE IT'S JUST A
POLYMORPHISM.
IT HEAP HELPING DIAGNOSING.
WEI A LONG WAY FROM WHY CERTAIN
MUTATIONS CAUSE COLON CANCER,
IN WOMEN IT'S OVARIAN OR BRAIN
CANCER.
THERE'S A LONG WAY TO GO.
UNDERSTANDING MOLECULAR
MECHANISMS EMPOWERS US TO FIGHT
THE WAR AGAINST CANCER.
I SWITCH GEARS NOW FOR THE NEXT
TEN OR FIFTEEN MINUTES ON THE
DNA PRELIMINARIES OF
TRANSLESION SYNTHESIS.
DNA ARE NOT ALWAYS PERFECT.
YOU NEED SPECIAL PRELIMINARIES
TO B BYPASS ROAD BLOCKS.
WHEN ROGER WOODGATE FOUND A NEW
ONE, HE APPROACHED ME.
WE TALK SOLVED THE STRUCTURE.
WEI TELLING YOU A GOOD
PRELIMINARY SET, MAKES VERY FEW
ERRORS AND MAKES MANY ERRORS.
THE DIFFERENCE IS IN THE ACTIVE
SITE.
HERE YOU CAN SEE AN ACTIVE SITE
REPRESENTED BY TWO BLUE SERIES,
HERE YOU CANNOT SEE IT BECAUSE
THE GOOD PRELIMINARIES IS
HIDDEN INSIDE.
IT'S MUCH MORE ENCLOSED AND
VERY MUCH MORE STRONGLY
DISCRIMINATING AGAINST THE
ROUND BASE OR NOT GOOD
TEMPLATE.
THIS IS NOT EXACTLY TELLING US
HOW HUMANS WORK.
IN HUMANS, PEOPLE WHO HAVE
MUTATIONS IN THAT GENE HAVE
HIGH SENSITIVITY TO UV LIGHT.
AND NORMALLY YOU KNOW WE GO
OUT, ENJOY THE SUN, BECAUSE IT
HELPS US STRENGTHEN THE BONES,
CONVERTING CHOLESTEROL TO
VITAMIN D.
BUT THOSE PEOPLE WHO HAVE
DEFICIENCY TO REPAIR UV LESION,
THEY ARE SENSITIVE AND HAVE
HIGH INCIDENCE OF SKIN CANCER.
WHAT UV DOES TO US, ULTRA
VIOLET, THE ADJACENT BASE
POINTS, EACHTHREE MONTHS,
CAN SERVE AS A TEMPLATE FOR DNA
PRELIMINARIES.
THEY GET CROSS LINKED, TWO
BASES LINKED TOGETHER LIKE A
SIAMESE TWIN.
20% OF TWO BASES BECOME 90
DEGREES TO EACH OTHER, SIX-FOUR
PRODUCT.
BECAUSE THIS IS A MORE
DIFFERENT FROM NORMAL, ACTUALLY
THE REPAIR OF EXCISION, A BERT
SUBSTRATE TO BE KHRAOEF CLEAVED OFF.
IT'S HARDER TO BE REMOVED BY
DNA REPAIR, THE MOVING CREW.
THERE ARE EIGHT GENETIC GROUPS
THAT MUTATIONS CAUSE PEOPLE TO
BE OVER SENSITIVE TO SUNLIGHT.
SEVEN OF THEM ARE INVOLVED IN
THIS REPAIR PATHWAY TO REMOVE
THE LESION, AND THE DISEASE,
ABCDEFG, ALL GREENS INVOLVED,
SYNTHESIZING.D
THE ROLL OF THAT, IN THE SELL
CYCLE, DURING THE SYNTHESIZED
TIME, IT'S VERY DANGEROUS,
CUTTING THE DNA TO PIECES.
SO WE NEED A SPECIAL
PRELIMINARY IN HUMANS TO BYPASS
ONE, TWO, THREE, FOUR STEPS,
INSERTION BASED, EXTENDING IT.
IT'S BEEN SHOWN THE MOST
FAITHFUL AND MOST EFFICIENT IN
CROSSING THE BARRIER OF THE
DIMER.
THERE ARE A LOT OF
PRELIMINARIES, THEY NEED
SIGNALS TO RECRUIT THE CELLS,
SPECIAL PRELIMINARIES TO THE
REPLICATION FORECAST.
IT'S IMPORTANT FOR PCNA WHEN
CELLS ENCOUNTER SUSTAINED UV
LESIONS, AND PCNA, IT CAN
RECRUIT THE SPECIAL
PRELIMINARIES TO BYPASS THIS
ROAD BLOCK.
AND IT GOES THROUGH FOUR STEPS
OF SYNTHESIS AND HANDS OVER THE
FINE REPLICATION FORK BACK TO
THE ABOUT PRIMARIES.
SO WE WERE INTERESTED.
BECAUSE HUMAN PRO TEEN I PROTEIN IS MORE
DIFFICULT TO GETA POST DOC AND
THEY SPENTRN, MARK, HE THEY
TWO ON OR THREE MONTHS.
THE PROTEIN DNA IN TRACTION
WASN'T THE NATIVE ENTER ACTION
BECAUSE IT DISTORTEDTION
INTERACTION, CATALYTICS.
I'LL SHOW YOU THE DIFFERENCE
BETWEEN NOT SO PERFECT DNA
COMPLEX AND PERFECT DNA
COMPLEX.
THE PROTEIN MOVES SUBTLY.
BUT TO GET THE RIGHT STRUCTURE
WE SPEND ANOTHER TWO YEARS AND
THAT'S A TEAM OF A POST DOC AND
STUDENT, YEARS OUT.
THEY SPENT TWO YEARS,
ENGINEERING NEW CRYSTAL LADDERS
SO WE CAN SEE WHEN IT'S IN THE
CATALYTIC MOLD, SHOWING HOW IT
ACCOMMODATES THE DIMER IN THE
ACTIVE SITE, NOT MAKING ANY
ERRORS.
NORMAL PRELIMINARIES WOULDN'T
TAKE TWO BASE PAIR INTO AN
ACTIVE SITE.
BY HAVING THIS IT WON'T BE ABLE
TO CLOSE.
AND THEY SOLVED THREE MORE
STRUCTURES OF THE LESION WITH
DNA, EACH STEP OF BYPASS,
CROSSING THIS PHASE, THE NEXT
PHASE, THE NEXT PHASE.
I WON'T BORE WITH YOU THE
STRUCTURES BA BECAUSE THEY LOOK
QUITE SIMILAR.
ISWING YOU HERE HOW THE DNA
UP SUPERIMPOSED ON NORMAL DNA,
THEY LOOK VERY NORMAL.
ABOUT FIVE OR TEN MINUTES AGO I
WAS TELLING YOU A GOOD DNA IS
LIKE A GOOD SPRING, THE LESION
IS A BROKEN SPRING, THE REPAIR
PROTEIN HERE WHAT THE BROKEN
SPRING LOOK LIKES.
WITH A UV LESION BEING REPAIRED
BY THE REPAIR PROTEIN, IT WILL
EXPLOIT THIS FLEXIBILITY AND
THE IN BENT DNA.
SOMEHOW WHEN IT'S COMPLEX WITH
THIS LESION BYPASS DNA, THE DNA
ACTUALLY WAS ABLE TO KEEP DNA
STRAIGHT.
THE REASON THAT IF YOU ARE
BUILDING A WALL YOU WANT THE
BRICKS LAYING SCARILY ON TOP OF
ANOTHER, IF THEY ARE LEANING
OVER YOU CAN'T BUILD MORE.
THEY ARE USING EXTENSIVE
PROTEIN AND DNA INTERACTIONS TO
KEEP THIS DNA NORMALLY THAT'S
BENT STRAIGHT FOR THE CHEMISTRY
TO TAKE PLACE.
WE LIKEN THIS AS A MOLECULAR
SPLINT.
THE PROTEIN PRELIMINARIES WILL
KEEP THE DNA STRAIGHT IN ORDER
FOR DNA ISN'T 'TIS T SYNTHESIS TO OCCUR.
AT LEAST FIVE MUTATIONS THAT
MPV PATIENT, THE PIGMENTOSUT
PATIENTSES, THE MUTATIONS
AFFECT THE STURDINESS.
THIS SUPPORTS THIS PROTEIN
BACKBONE.
IN THE LAST COUPLE SLIDES I
WILL TURN THE ATTENTION TO THE
APPLICATION OF THIS STUDY OF
THIS SPECIAL POLYMERASE.
IT'S GOOD TO HELP US TO GUARD
AGAINST THE LESION CAUSED BY
UV, BUT IT PLAYS A NEGATIVE
ROLE IN TREATMENT OF CANCER
PATIENTS, IT'S BROADLY USED
CHEMOTHERAPY AGENT.
IT CROSS-LINKS, THE THYMINE
DIMERS HERE, IT CROSS-LINKS TO
ADJACENT, AND KILLS CANCER
CELLS BECAUSE SO MANY OF THE
LESIONS HERE, THE CELL WILL BE
STOPPED IN REPLICATION,
FAST-DIVIDING CANCER CELLS.
IT TURNS OUT THAT THE STUDY
BASED ON CANCER-TREATED
PATIENTS SHOWS IF YOU HAVE HIGH
LEVELS OF EXPRESSION LEVEL,
SHOWN HERE, THIS CURVE, THE
SURVIVING TIME IS MUCH SHORTER
THAN IF YOU HAVE LOW EXPRESSION
LEVEL BECAUSE IT PROBABLY
BYPASSES, CAUSING THE LESIONS,
LIKE UV LESIONS.
IN STAGE THREE DISEASE THE
DIFFERENCE IS EVEN MORE SEVERE.
IF YOU HAVE LOWER, PATIENTS
RESPOND MUCH BETTER TO THE
TREATMENT AND LIVE LONGER.
AND BIOCHEMICALLY, OTHER GROUPS
AS WELL AS US HAVE SHOWN IT CAN
BYPASS LESION EFFICIENCILY
INSERTING AGAINST 80% OR 85% OF
INSERTING AGAINST NORMAL
TEMPLATE.
IT DROPS A LITTLE WHEN IT GOES
ON THE NEXT BASE AND DROPS MUCH
FURTHER WHEN IT'S ON THE THIRD
AND FOURTH.
THE FIRST TWO ARE THE HARDEST
ONES TO GET BY THEMSELVES.
SO WE WERE LOOKING FOR WAYS TO
INHIBIT IT AND COMBINE
PLATINUM-BASED TREATMENT, THE
PATIENT WILL PROBABLY RESPOND
BETTER WITH LOWER DOSAGE AND
SURVIVE LONGER.
AND IT WAS ALL IN THE TALK,
PEOPLE IN ROCKVILLE, THE DRUG
SCREENING GROUP AND ALSO ROGER
WINGATE TALKING ABOUT IT, BUT
WE FIND SOMETHING IN THE
CRYSTAL STRUCTURE, SO WE WERE
CRYSTALLIZING WITH PRETTY HIGH
CONCENTRATION INCOMING, AND WE
FOUND A SECONDE SECOND BINDING SITE
AWAY FROM THE ACTIVE SITE.
WE WERE KIND OF BOTHERED BY
THIS DENSITY, HOW TO GET RID OF
IT.
IF WE STOP THE CRYSTAL WITH NO
INCOMING NUK, AND WHAT'S MORE
INTERESTING IS HERE, WE FIND
NORMALLY WHEN YOU INCREASE THE
SUBSTRATE CONCENTRATION, THE
ACTIVITY GOES UP.
WITH THIS, WHEN WE INCREASE 50
MICRO MOLAR, I'M GETTING
INHIBITUS.
INHIBITION CAN BE REMOVED WITH
THE BASE OF THE INCOMING, IF WE
MUTATE THE TRYPTOPHAN, NOT
CONSERVED IN THE Y FAMILY
POLYMERASE, NOW THE ACTIVITY WE
THINK BECAUSE THE INTERACTION
WITH DNA IS AFFECTED, BUT IT'S
NO LONGER BEING INHIBITED.
SO THIS MAY BE A GOOD TARGET
FOR FINDING NEW DRUGS TO
INHIBIT AND USE FOR CANCER
THERAPY.
I'LL CONCLUDE MY TALK LEER, SO
I SHOWED YOU ABOUT DNA LESIONS
WHICH CAN BE CAUSED BY EXTERNAL
EFFECTS OR ENVIRONMENTAL
AGENTS, OR JUST BECAUSE THEY
MAKE AN ERROR LEADING TO
INCREASED FLEXIBILITY IN DNA.
IT'S USED BY BOTH REPAIR
ENZYMES, ASSISTED BY ATP FOR
REPAIR, OR FORTIFIED BY SPECIAL
PRELIMINARIES TO CARRY OUT
TRANSLESION DNA SYNTHESIS.
IN BOTH PROCESS, THE OUTCOME IS
PREVENT THE MUTATION AND REDUCE
THE CANCER INCIDENTS.
THE SITE MAY BE LEADING US TO
THINK ABOUT DEVELOPING DRUGS
FOR INHIBITION AND HELPING
EFFECTIVENESS OF CANCER
TREATMENT.
SO I'VE ACKNOWLEDGED, I TALK
ABOUT THE WORK AND HERE IS THE
CURRENT GROUP.
WE HAVE PICTURES TAKEN TOGETHER
WITH MARTY'S GROUP, WE HAVE
EXTENDED LABS, A LOT OF
PEOPLE'S WORK, I'M NOT
MENTIONING HERE BECAUSE THEY
ARE STILL IN THE WORKING WITH
SOME LARGER COMPLEX.
I MENTIONED BY FORMER PEOPLE,
ALSO FORMER POST DOC STUDENTS,
COLLABORATORS, AND WE
COLLABORATED WITH' IN JAPAN, A LOT OF
WONDERFUL CLACK LATORS COLLABORATORS, MAN Y
YOU RECOGNIZE FROM NIH,
SUPPORTED BY INTRAMURAL
FUNDING.
LASTLY BUT NOT LEAST I WANT TO
ACKNOWLEDGE LABORATORY OF
MOLECULAR BIOLOGY AT NIDDK,
MANY ARE HERE.
I FEEL SORT OF HIGHLY PRESSURED
AND NERVOUS BECAUSE I'M JUST
REPRESENTING OUR GROUP, BECAUSE
ALL OF THEM, EVERYONE OF THEM
HAVE A WONDERFUL STORY TO TELL,
IT'S A GREAT GROUP TO LONG TO.
THE SENIOR MEMBER INSPIRES ME
BY DOING THE SIGNS THE WAY THEY
DO, THE ACTION, THEY SHOW
WHAT'S IMPORTANT, AND JUST
GREAT FUN TO BE A MEMBER OF
THIS FAMILY, AND ALSO BELONG TO
OTHER GROUPS, VERY SUPPORTIVE,
I HAVE A LOT OF POSITIVE AND
INTERESTING INTERACTIONS WITH
MEMBERS AND ALSO THE SOCIETY OF
CHINESE SCIENTISTS IN AMERICA,
NIH, WE HAVE SPORADIC
INTERACTION AND VERY SUPPORTIVE
IN FORMING A LOT OF
CONVERSATIONS AND HOPEFULLY
PRODUCTIVE COLLABORATIONS IN
THE FUTURE.
THANK YOU ALL.
[APPLAUSE]
>> QUESTIONS?
>> YES.
GREAT TALK.
MY QUESTION IS WHE HOW IT INTERACTS
WITH MutS?
>> YOU MEAN THE LESION SEARCH?
>> YES.
>> RIGHT.
IT CANNOT BE SORT OF SLIDING ON
DNA FOREVER.
IT'S ONLY SLIDING ON VERY SHORT
DISTANCE.
IT HAS TO BE -- IT'S A COMBINED
3D, 2D SEARCH, THE ASSOCIATION
IS IMPORTANT.
AND THEN WE'LL HIT ANOTHER
SPOT.
>> THANK YOU.
>> YES, USUALLY COMBINED 1D AND
3D MOVEMENTS IN THE NUCLEUS.
>> ALL RIGHT.
HAVE THE STRUCTURE GIVEN YOU
ANY HINT AS TO HOW THE COMPLEX
CAN TELL THE OLD FROM THE NEW
DNA STRAND?
>> RIGHT.
IN BACTERIA, E. COLI, IN SOME
IT'S CLEAR BECAUSE THE TEMPLATE
IN E. COLI IS METHYLATED, THE
DAUGHTER STAND IS NOT
METHYLATED.
THE NUCLEUS WILL BE CUTTING THE
METHYLATED STRAND.
ANY OTHER BAC BACTERIA IT'S NOT
CLEAR.
IN HUMANS THE CONSENSUS,
MOVES, COM POLYMERASE DOESN'T
FAST, THE TREN STRAND IS RECOGNIZED.
THIS IS STILL -- THE JURY IS
STILL OUT BECAUSE WE HAVEN'T
FOUND THE NUCLEUS CLEAVING THE
DAUGHTER STRAND SPECIFICALLY,
IT'S MORE RECENT THAT MutL
CAN CAUGHT DAUGHTER STRAND, YOU
HAVE A THREE-WAY INTERACTION,
THE PCNA, THE LOADING CLAMP,
WILL BE LOADED IN CERTAIN WAYS
RELATIVE TO THE PRIMARY STRAND.
I NEED HELPERS TO DIFFERENTIATE
TWO-STRAND.
>> SO I'M AFRAID THIS IS GOING
TO BE A RATHER ARCANE QUESTION.
BUT IT COMES FROM MY INTEREST
IN HETERODUPLEX DNA,
RECOGNIZING A CC MISMATCH IS
ONE OF THE MOST DESTABILIZING
OF THE DNA DUPLEX.
YET IT IS THE MISMATCH THAT IS
NOT RECOGNIZED.
>> NOT RECOGNIZED WELL BY
MutS, YOU KNOW WHY?
>> I DON'T KNOW WHY.
THAT'S WHY I'M ASKING.
>> THE ANSWER IS TWO SIDES OF
THE COIN.
POLYMERASE DOESN'T MAKE C.C.
MISMATCH, BECAUSE CC IS SO
UNSTABLE, THE POLYMERASE
DOESN'T MAKE IT MUCH.
IT'S THE SAME SIDE.
TWO SIDES OF THE COIN.
IT DOESN'T MAKE MUCH.
IT'S HUMAN MADE.
>> BUT IN TERMS OF THE
MOLECULAR MECHANISM, IN TERMS
OF WHAT YOU SAID IN TERMS OF
PROBING THE DNA STRUCTURE FOR
FLEXIBILITY, I WOULD HAVE
EXPECTED IT TO BE THE MOST
FLEXIBLE.
>> IT'S SOME WHAT DIFFERENT.
NO, IT'S DIFFERENT.
>> OKAY.
>> YEAH, SO NORMALLY THE BASE
WOULD BE STACKED WEAKER, LIKE
DOING THIS.
BUT IT'S SO BAD MOST LIKELY
THEY ARE PUSHED OUT, AND
ADJACENT BASE BECOMES STACKED.
>> OKAY.
>> THAT BECOMES MORE STABLE.
AND BECAUSE THE BASE PUSHED
OUT, NOT WELL RECOGNIZE THE BY
MutS BUT BUT OTHER PATHWAYS.
>> RIGHT.
>> THAT WAS BEAUTIFUL.
SO WITH THE STORY WHERE YOU
SHOW THE SPLINT STRAIGHTENING
THE DNA, BUT BEFORE POL-A GET
THERE, THE DNA IS BENT
>BENT.
>> THE DNA IS NOT ALWAYS BENT.
THE INCREASED FLEXIBILITY MEANS
IT ISN'T -- NORMALLY THE DNA --
IF IT'S HALF A LESION, IT DOES
THIS.
IT COULD BE STRAIGHT.
IT'S VERY LITTLE.
SO THE POLYMERASE CAPTURED IT,
WENT STRAIGHT AND CAPTURED IT
STRAIGHT.
THAT'S MOST OF THE SAME.
PROBABLY 95% OF THE TIME IT'S
NOT INDUCED TO FIT.
THE CONFIRMATION IS RONALD
THERE IT'S NOT STABLE AND GETS
TRAPPED.
>> BEFORE I THANK WEI, I WANTED
TO MAKE A COUPLE ANNOUNCEMENTS
WHILE I HAVE THE CAPTIVE
AUDIENCE.
THE FIRST IS THAT THE WEDNESDAY
AFTERNOON LECTURE NEXT WEEK IS
ON THURSDAY.
SO CHANGE YOUR CALENDARS
ACCORDINGLY.
SECONDLY, WE HAD ANOTHER
INTERESTING EVENT HERE AT NIH,
WHICH WAS SPONSORED BY THE
JOURNAL OF SCIENCE, A WEB THAT
SO CALLED SUPER-RESOLUTION
IMAGING, NOT THE RESOLUTION OF
X-RAY CRYSTALLOGRAPHY BUT
PRETTY GOOD.
NIH TODAY RECORDED DISCUSSION
WITH HARRY SHROFF, AND CLAIRE
WATERMAN, AND THAT WEBINAR WILL
TAKE PLACE OFFICIALLY ON
WEDNESDAY, FEBRUARY 29th AT
NOON.
SO IF YOU'RE INTERESTED IN
CHECKING IN, I THINK YOU MIGHT
FIND THAT AN INTERESTING EVENT.
SO I JUST WANTED TO SAY THAT IN
DISCUSSING THIS LECTURE BEFORE
WEI GOT ON THE PODIUM, SHE TOLD
ME SHE FELT THE BURDEN OF
REPRESENTING THE LABORATORY OF
MOLECULAR BIOLOGY WHICH IS ONE
OF THE GEMS O OF THE NIH, THE
NIDDK AND WOMEN SCIENTISTS,
CHINESE-AMERICAN SCIENTISTS,
AND SHE WAS WORRIED SHE WOULD
ACQUIT HER IN THE BEST POSSIBLE
WAY AND I CAN EYE SUR ASSURE YOU,
YOU'VE DONE A MAGNIFICENT JOB.
THANK YOU VERY MUCH.
[APPLAUSE]