>> WELCOME, EVERYONE. HOPEFULLY EVERY ONE IS CONNECTED. KEN KRAEMER AND WILL BOHR ARE AWAY, SO IF ANYONE REMEMBERS TWILIGHT ZONE DON"T ADJUST YOUR TV SET. I'M NEITHER OF THOSE TWO. I'M A JOHN A DERMATOLOGIST AT NCI AND I'VE WORKED WITH KEN FOR DECADES. WE ARE VERY PREASED TO HAVE MICHAEL LIPTON OWN BUILDING 37, WHO IS THE DEPUTY CHIEF OF THE LABORATORY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY. HE HAS CHOSEN A GREAT TITLE FOR HIS TALK, CHOOSING THE RIGHT PARTNER GETTING TO THE RIGHT OUTCOME IN MIOSIS AND WE WILL TRY TO GET THE SLIDES RUNNING. AND -- >> OKAY, GREAT. SO I'D LIKE TO THANK ALL OF YOU, HOWEVER MANY OF YOU THERE ARE FOR JOINING US. IT'S HARD TO TELL. NORMALLY IN A TALK LIKE THIS I WOULD SAY IF YOU HAVE ANY QUESTIONS, JUST RAISE YOUR HAND AND JUMP UP AND I'LL BE HAPPY TO ANSWER BUT RAISE YOUR HAND ALL WANT, I WON'T BE ABLE TO SEE. SO WHAT I AM GOING TO TALK ABOUT TODAY IS WORK THAT MY LAB HAS BEEN WORKING ON USING MIOAT -- MIOTIC RECOMBINATION TO STUDY FUNDAMENTAL MECHANISMS OF HOMO HOMO/LOGOUS RECOMBINATION. SO I'D LIKE TO START OUT JUST WITH A BRIEF OVERVIEW AND JUST TO POINT OUT THAT DOUBLE STRAND S CONSTITUTE A MAJOR THREAT TO THE IN/TEGRITY OF THE GENOME. AND SO HAVE VARIOUS STRATEGIES TO /REPAIR THESE BREAKS. BUN THAT GETS A LOT OF ATTENTION IS UNTEMPLATED REPAIR OF WHAT'S CALLED NON-HOMO/LOGOUS END JOINING, WHICH IS BASICALLY A FORM OF LIGATION. ALTHOUGH THIS DOES REPAIR THE BREAK, IT COM COMPROMISES IN/E OF THE GENOME. SO EVEN UNDER BEST CIRCUMSTANCES, THERE IS MUTATION INVOLVED, USUAL RIBASES ARE GAINED OR LOST LOST, AND OF COURSE, BECAUSE IT'S JUST /AA LIGATION REACTION, THERE IS ALWAYS THE RISK OF GENOME REARRANGEMENT THROUGH TRANSLOCATION. BY CONTRAST, HOMO/LOGOUS RE RECOMBINATION, WHICH AS /AA FIRST STEP, INVOLVES THE TWO BACK OF ENDS TO EXPOSED SINGLE STRANDS, WHICH ARE THEN USED TO INVADE A MOM OLOGOUS DUPEEX DNA AND DNA SIN /TYNTHESIS. BECAUSE IT'S TEMPLATED, REPAIRS THE DOUBLE STRAND BREAK WITH RELATIVE ACCURACY. AND THESE ARE THE TWO PRODUCTS THAT ONE CAN GET. A NON-CROSS /KWROEOVER AND CROSSOVER. AND ALTHOUGH SOMETIMES HOMO/OL HOMOLOGOUS RECOMBINATION DOESN'T -- DOESN'T GET THE RESPECT IT DESERVES. A COMPARISON IN MUTATION RATE WITH ESTIMATED DO YOU BELIEVE STRAND BRACE INDICATE THAT HOMO HOMO/OL/KPW*US RECOMBINATIONS ARE THE PREFERRED MODE OF DNA DAMAGE REPAIR BAIT LEAST A COUPLE /OF ORDERS OF MAGNITUDE. SO THAT'S IN MITT /OOTIC CELLS. IN MI/OOS, I WHICH IS THE PROCESS BY WHICH A GENOME IS REDUCED TO 4 HAPLOID GAMETES, THE RECOMBINATION IS ABSOLUTELY ESSENTIAL. DO -- SO DURING THE PROCESS OF US MI/OEIOSIS AND I'VE SHOWN HERE A GENOME, ONCE THE CHROMOSOMES REPLICATE, THEY PAIR AND AS YOU WILL SEE UNDER GO RECOMBINATION. AND THAT PARING IS ESSENTIAL FOR THEIR SEPARATION DURING THE FIRST MYOTIC DIVISION, WHICH IS REALLY WHAT REDUCES THE DIPLOID GENOME TO HAPLOID. MANY -- NOT ONLY ARE THE STEPS OF HOMO/LOGOUS RECOMBINATION ESSENTIAL TO BRING THE TWO COPIES OF /AA GIVEN CHROMOSOME TOGETHER, BUT THE CROSS /KWROEOVER PRODUCT OF MYOTIC RECOMBINATION ARE ESSENTIAL TO CONNECT THE TWO HOMO/OLOS --OGS /STKWHR-RGS BY A ALLOWING THEM TO BE ALIGNED, A ATTACHED CELL -- CORRECTLY TO THE SPINNEDLE AND BY PROVIDING A CONNECTION THAT ALLOWS TENSION, ALLOWS THE SPINNDLE TO SENSE THE THAT THE APPROPRIATE CONNECTION HAS BEEN MADE AND TO SEPARATE THE CHROMOSOMES PROPERLY. FOLLOWING THAT, A SECOND MITOSIS MITOSIS-LIKE DIVISION HAPPENS. TO -- SO RECOMBINATION PLAYS AN IMPORTANT ROLE IN TWO STAGES. THE EARLY STEPS OF HOMO/LOGOUS RE RECOMBINATION ARE CRITICALUAL FOR RECOGNITION AND RE/SKPAEURGT CROSS /KWROEOVER PRODUCT OF MYOTIC RE RECOMBINATION ARE ESSENTIAL FOR SEPARATION OF THIS /KWEURS DIVISION. SO YOU MIGHT IMAGINE AS /AA CON CONSEQUENCE, WHEN RECOMBINATION GOES WRONG DURING MY /OOSIS, THINGS GO BAD VERY RAPIDLY. AND IN FACT, IN THE ABSENCE OF RECOMBINATION OR IF RE RECOMBINATION OCCURS FROM THE WRONG PLACE, ONCE FAILURES TO SEPARATE HOMOLOGS PROPERLY AT MY OSIS ONE, LEADING TO EITHER THE ABSENCE OF CHROMOSOMES OR EXTRA COPIES OF CHROMOSOMES AMONG THE GAMETES, SUBSEQUENT CHROMOSOME IMBRILLIANCE IN ZYGOTES AND AS A RESULT, BAD CONSEQUENCES. IN HUMAN POPULATIONS, CHROMOSOME IMBALANCE IN DYING OATES IS A CAUSE OF /AA SIGNIFICANT CAUSE OF INFERTILITY, AND IN ADDITION, OF COURSE, THROUGH CHROMOSOME 21 TRISOMEING IS /THE LEADING CAUSE OF BIRTH DEFECTS IN NEWBORN /PHREPBL TARDATION. SO IT'S REALLY IMPORTANT FOR ORGANISMS TO GET RECOMBINATION RIGHT AS THEY GO THROUGH MY /OOSIS. AS /AA CONSEQUENCE BECAUSE OF THIS NEED FOR RECOMBINATION BETWEEN HOMOLOGS AND IN PARTICULAR BETWEEN CROSSOVERS BETWEEN HOMOLOGS, CELL USE A VARIETY OF STRATEGIES TO ENSURE THOSE OUT OUTCOMES. FIRST OF ALL /SKWR-RB, INSTEAD OF RELYING ON ACCIDENTAL DNA DAMAGE, THEY PURPOSELY FORM DOUBLE STRAND BREAKS IN THEIR GENOME. IN EASIEST A COUPLE /OF0 HUNDRED BREAKS PER NUCLEUS UP TO SEVERAL THOUSAND DOUBLE STRAND BREAKS FOR -- PER NUCLEUS. THESE DOUBLE STRAND BREEZY ARE FORMED BY THE END /-OF THE NUKE LASE. AFTER DOUBLE STRAND BREAKS ARE FORMED, SO USE A STRATEGY THAT PREVENTS RECOMBINATION BETWEEN SISTER CHROMAT AD -- CHROMATIDS, AND INSTEAD DIRECT RECOMBINATION TOWARDS RECOMBINATION BETWEEN HOMOLOGS.^ AND THEN ONCE THE INITIAL PHASES OF RECOMBINATION OCCUR, THE CELLS USE A VARIETY OF STRATEGIES TO PROMOTE CROSS CROSSOVER FORMATION, IN ADDITION TO NON-CROSS /KWROEOVER FORMATION. SO IN THE MITT /OOTIC CELL CYCLE, THE VAST MAJORITY OF HOMO/LOGOUS RECOMBINATION PRODUCTS ARE NON- NON-CROSSOVER. IN THE MYOTIC CELL CYCLE, CROSS THE /KPWROEFRZ ARE PROMOTED, FOR EXAMPLE IN /AA YEAST ORGANIZSM, ABOUT HALF OF THE PRODUCTS ARE CROSSOVERS. SO THIS INTERHOMOLOG CROSSOVERS ARE PROMOTED THROUGH A VARIETY OF STRATEGIES, MOST PROMINENT ARE MI/OEIOSIS-SPECIFIC BIOCHEMICAL ACTIVITIES THAT SPECIFICALLY PROMOTE ARE HOMO/LOGOUS COMBINATION AND IN ADDITION SPECIALIZE MYOTIC CHROMOSOME STRUCTURES ARE PUT DOWN BY MY OS OSIS AND THESE HOMOLOG RE RECOMBINATION AND STABILIZE RE REBEGCOMBINATION AND ULTIMATELY PROMOTE CROSS /KWROEOVER SPOT.^ AS I SAID, MY LAB STUDIES MYOTIC RE RECOMBINATION, USES MY /OOSIS AS A COMBINATION. TO STUDY HOMO/LOGOUS WE DO SO FOR /AA VARIETY OF REASONS. FIRST OF ALL /SKWR-RB, ALTHOUGH YEAST IS VERY SLOW, IT HAS FUNCTION /AAL HOMOLOGY WITH LARGER EU/KKARYOTES IN BOTH THE LARGER RECOMBINATION PROTEIN, THE MYOTIC, THE CHROME CHROMOSOME STRUCTURAL PROTEINS THAT ARE LAID DOWN AND IN TERMS OF MI/OEIOSIS-SPECIFIC RE RECOMBINATION CHROMOSOME STRUCTURES. A YEAST IS AN ORGANISM THAT IS THE CHARACTERIZED BY WHAT I CALL GOOD GENETICS AND GOOD GENOMICS. ITS GENOME IS QUITE LIKELY THE BEST CHARACTERIZED OF ALL EU EUKARYOTES AND IT'S EASILY MANIPULATE SOD WE CAN MAKE CHANGES TO THE GENOME AT WILL. IT ALSO HAS THE ADVANTAGE OF HAVING VERY SMALL KENONE AS /AA CONSEQUENCE. RECOMBINATION IS FREQUENT ON A EVENT PER KB BASIS AND BECAUSE OF THIS HIGH DENSITY REF COMBINATION /SKWRAOERPBGTS IT'S /KPWRAOEFRZ TO SEE THEM. AND FINALLY, EASIEST -- YEAST HAS THE ADVANTAGE OF BEING ABLE TO INDUCE MY /OOSIS IN SIYNCHRONOUS CULTURES IN /SKPHR-BGD THAT, COMBINED WITH A HIGH FREQUENCY OF RECOMBINATION, ESPECIALLY, ALLOWS US TO FOLLOW THE MOLECULAR EVENTS OF MYOTIC RE RECOMBINATION IN REALTIME AT THE DNA LEVEL. SO JUST TO GIVE YOU A QUICK EXAMPLE OF HOW WE GO ABOUT DOING THIS. PLEASE DON'T BOTHER WITH THE DETAILS OF THIS SLIDE. YOU'LL JUST HAVE TO TRUST ME. BUT I JUST BANT /TO SHOW TO POINT OUT THAT USING -- LOOKING AT DNA FROM MYOTIC CELLS, WE CAN DETECT MANY OF THE STEPS OF MYOTIC RE RECOMBINATION. SO WE CAN DETECT DOUBLE STRAND BREAKS, COMING UP HERE. AND GOING AWAY AS WE SEE REBEGIN RECOMBINANT PRODUCTS. USING ANOTHER CELL SYSTEM, WE CAN DETECT RECOMBINATION INTER INTERMEDIATE IN PARTICULAR, IN -- THIS CHARACTERISTIC DOUBLE JUNCTION JOINT MOLECULE AND THESE ARE COMING UP HERE. AND FINALLY USING YET ANOTHER SET /OF RESTRICTIONS I -- WE CAN DETECT BOTH THE NON-CROSS NON-CROSSOVER AND THE CROSSOVER PRODUCT. , MYOTIC RECOMBINATION. SO BASICALLY, BY LOOKING AT THE DNA, WE CAN FOLLOW THE MOLECULAR EVENTS OF MY /OOSIS AND CAN CORRELATE THOSE WITH THE OTHER EVENTS THAT ARE OCCURRING, FOR EXAMPLE, AT THE CITE LODGEAL AND CHROMOSOMAL STRUCTURAL LEVEL. SO WORK IN /AA NUMBER OF OTHER LABS, A LARGE NUMBER OF OTHER LABS, THEY ARE LISTED AT THE BOTTOM OF THIS SLIDE -- HAVE GIVEN US THE FOLLOWING PICTURE OF HOW RECOMBINATION IS INT INTEGRATE ED INTO THE MYOTIC CELL CYCLE. SO AFTER DNA REPRESELICATION, THE FIRST THING THAT HAPPENS IS THAT A SET /OF PROTEINS ARE LAID DOWN THAT FORM A MY /OOSIS-SPECIFIC CHROMOSOME AXIS AND THIS AXIS COORDINATES CHROMOSOMES, SISTER CHROMATIDS DUE TO A SERIES OF BASIC LOOPS THAT ARE TETHERED TO THE AXIS. DOUBLE STRAND BREAK FORMATION OCCURS WHETHER A NUMBER OF OTHER ACCESSORY PROTEINS ARE RECRUITED NOT TO THE DOUBLE STRAND BREAKS BUT TO THE AXIS ITSELF. AND THEN BREAKS FORM WHEN TARGET SEQUENCES IN THESE LOOPS ARE RE RECITED TO THE AXIS AND CUT SCROLL. SUBSEQUENT TO DOUBLE STRAND BREAK FORMATION, THE BREAK IS RECOGNIZED BY THE CHECKPOINT KINASES AND THESE CHECKPOINT KINDS /OF DNA DAMAGE RESPONSE KINASES AND THESE KINASES FOSS PHOSPHORYLATE AS PROTEIN, RE RECRUIT YET ANOTHER DOWNSTREAM KINEASE AND THIS SETS UP A RE RECOMBINATION SLOW ZONE THAT LIMITS THE CHROMAT ID COMBINATION COMBINATION. SO LEAVING DOUBLE STRAND BREAK ENDS FREE TO ENCOUNTER THE HOMO HOMOLOG OUTSIDE OF THIS RE RECOMBINATION ZONE. MUCH OF THIS WORK IS WORK THAT WAS DONE BY NANCY HOLLINGS WORTH AT STONE /KWRY BROOK AND YOU MAY HAVE HEARD HER AT AN EARLIER TALK IN THIS VENUE EARLIER THIS /KWRAEYEAR. SO THIS IS BASICALLY BY MODIFICATION OF THE AX /KPWREUS IN RESPONSE TO DOUBLE STRAND BREAKS, THE CELL SETS APE BAR BARRIER TO SISTER CHROMAT /KPWREUD RECOMBINATION AND THERE BY PROMOTES RECOMBINATION TO INTERHOMOLOG EVENTS. THESE ARE HOMOLOG EVENTS FORM EARLY STRAND INVASION INTERMED INTERMEDIATE AND THESE WHICH ARE QUALITY STABLE ARE BASICALLY USED IN ORDER TO CO-LOCALIZE HOMOLOGS AND TO PULL THEM TOGETHER, CULMINATEING IN THE A ASSEMBLY OF /AA MY /OEOSIS-SPECIFIC STRUCTURE CALLED THE SYNAPSEIC COMPLEX, WHERE HOMOLOGS, THESE PAIRS OF SISTER CHROMATIDS, ARE PAIRED END TO END BY A PROTEIN PROTEINACIOUS STRUCTURE THAT INCLUDES THE AXIS AND AT /THIS STAGE THREE THINGS HAVE HAPPENED TO THESE EARLY STRAND INVASION INTERMEDIATES. A LARGE NUMBER OF THEM HAVE BEEN TAKEN APART AND TURNED INTO MATURE NON-CROSS /KWROEOVER RE A, IN YEAST AN EQUAL NUMBER HAVE BEEN STABILIZED BY PROTEINS OF AN ASSEBLY WHICH I WILL REFER TO AS THE RECOMBINATION NODULE. THIS IS BASICALLY A VISIBLE BLOB OF PROTEIN THAT DECK CRATES THE COMPLEX. THESE PROTEINS STABILIZE THESE EARLY STRAND IN INVASION INTERMEDIATES AND PROMOTE THEIR MATURATION INTO DOUBLE /HOPLD JUNCTIONS. FINALLY, A MINOR SET ABOUT ABOUT ON THE ORDER OF T10% TO 20% -- I'M SORRY, I'VE GOTTEN AHEAD OF MYSELF. SO FOR EXAMPLE, ONE PAIR OF PROTEINS MSH 4 AND 5, FIREARM SORT OF CLAMP THAT BINDS TO THESE HOLIDAY JUNCTIONS AND STABILIZES THESE INTERMEDIATES PROTECTING THEM AND PREVENTING THEM FROM BEING TAKEN APART. IN ADDITION THO-- TO THESE TWO MAJOR PATHWAYS FOR MYOTIC RE RECOMBINATION THERE IS A MINOR PATH BYE THAT CONSTITUTES OH, ON THE ORDINARY -- ORDER OF TEN TO 20%, WHICH IS BASICALLY UN UNREGULATED IN THAT IT DOESN'T SHOW A STRONG PARTNER BIAS. THEY'RE LOT /OF INTERSISTER RE RECOMBINATION INTERMEDIATES FORMED AND THEN THE I SHOULD -- YOU CAN'T SEE THEM HERE. I MADE IT TOO SMALL. BUT THESE SOMETIMES CAN CONTAIN CONTRIBUTIONS FROM NOT TWO CHROMOSOMES BUT THREE OR FOUR CHROMOSOMES SPOT.^ THESE ARE ALL PATH WWAY-INTERMEDIATE. OKAY. THEN THIS STAGE OF -- AT /THIS STAGE WE'RE -- WHERE SIGNIFICAYNAPTIC COMPLEX IS FORMED, WE'VE GOT MATURE, HOLIDAY JUNCTION INTERMED DOUBLE /SKPWRATS A SMALL FRACTION OF INTERMEDIATE AND THEN IN GREATEST AT THE END OF PACKAGING PACKAGING, A TRANSCRIPTION FACTOR TURNS ON T. D. 5 KINASE. AND POLL-LIKE KINASE CAT LIES NUMBER OF EVENTS. FIRST OF ALL /SKWR-RB, IT PROMOTES THE DIS ASASSEMBLY OF THE SIGNIFICANYNAPTIC COMPLEX. I WON'T SHOW YOU THAT. SECOND OF ALL, IT ACTIVATES A MY OSIS WIARE-SPECIFIC HOLIDAY JUNCTION RE/SOSOLVE ASASE MADE UP OF THE MISMATCH REPAIR PROTEINS MLH 1 AND 3 AND XO1 AND THIS MI/OEIOSIS MEIOSIS-SPECIFIC RESOLVES THE OLIDAY JUNCTIONS SPECIFICALLY AS CROSSOVERS. FINALLY, DDC 5 ALSO ACTIVATES A SET /OF MITT /OOTIC RE/SOFSOLVE ASASES. IN YEAST THESE PROTEINS AND THESE ARE NUKE LASES THAT ARE ACTIVE DURING THE MITT /OOTIC CELL CYCLE. THESE MITT /OOTIC RE/SOFSOLVE AGENTS RE RE/SOSOLVE THESE MOLECULES AND THEY FORM BOTH CROSSOVERS AND NON- NON-CROSSOVERS. SO BASICALLY I'LL MOVE /OON FROM THERE. SO I AM GOING TO TALK ABOUT TWO /TPOPICS TODAY. THE FIRST /OF WHICH IS ADDRESSING -- ADDRESSES THE QUESTION OF WHAT'S RESPONSIBLE FOR CONTROLLING THE DECISIONS AS TO WHETHER AN -- A RECOMBINATION INTERMEDIATE >> A RECOMBINATION EVENT IS GOING TO FORM A NON-CROSSOVER, IS GOING TO FORM A RECOMBINATION NODULE-PROTECTED DOUBLE HOLIDAY JUNCTION THAT WILL BE RESOLVED IS A CROSS /KWROEOVER? OR IS GOING TO GO DOWN THIS SIDE PATH TWY FORM AN ABERRANT JOINT MOLECULE THAT WILL BE RESOLVED BY THE MITT /OOTIC. AND IT TURNS OOUT THAT THIS HELO HELOTASTE COMPLEX THAT PRAYS AN IMPORTANT ROLE IN GENOME STABILITY IN THE MITT /OOTIC CELL CYCLE ACTUALLY IS /THE CENTRAL ACTIVITY THAT CONTROLS THE CHOICE BETWEEN THESE,000 -- THREE PATHWAYS. AND THIS IS THE WORK OF -- IN THE LAB. THIS WORK HAS BEEN PUBLISHED MOST RECENTLY THIS SUMMER AND SO I WILL TOUCH UPON IT BRIEFLY BEFORE I GO ON TO THE SECOND TOP /KWREUIC. SO THE SECOND TOP /KWREUIC ADDRESSES THE QUESTION OF THE ROLE OF CHROMOSOME STRUCTURE IN INFLUENCEING MYOTIC RE RECOMBINATION. AND THE TAKE-HOME LESSON WILL BE IS THAT CHROMOSOME ENVIRONMENT ALLY WHICH RECOMBINA IS CRITICAL IN DETERMINING PATHWAY IS GOING TO BE USED. AND THIS IS THE WORK OF MEDY, A GRADUATE STUDENT BETWEEN MY LABORATORY AND LAB OF GOLDMAN AT THE UNIVERSITY OF SHEFFIELD. SO TO RETURN TO THE FIRST TOPIC, HOW S /T-FPTS 1 AND MRI 1, WHICH I WILL REFER S /T-FPTR, IN THE INTEREST OF TIME, CONTROL MYOTIC RECOMBINATION PATH WWAY CHOICE. SO JUST /AA LITTLE INTRODUCTION SO -- TO S /T-FPTR? IT'S COMPOSEED A HELOTASTE, SGS 1 WHICH IS HAM -- HOMO/LOGOUS TO THE MAM /MALIAN BLOOM, WHICH I AM SURE. YOU HAVE FAMILIAR WITH. BLOOM HAS PLAYED A VERY IMPORTANT ROLE IN MAINTAINING GENOME INTEGRITY AND BLOOM SYNDROME AGENTS WHO LACK FACILITATE SHOW PREMATURE AGE AG AG, GENOME INSTABILITY AND GET CANCER. BUT MOST IMPORTANT FOR THE PURPOSES OF THIS TALK, IS -- THEY SHOW ELEVATED MITT /OOTIC RE RECOMBINATION RATES. BLM AND SGS 1 ARE BOTH BOUND IN SE 3 AND ITS PARTNER PROTEIN RM RMI 1 AND IN MUTANTS, YOU CAN SLACKEN OR MUTANT SLACK SLAEN 3. AGAIN SHOW PREMATURE AGING. THEY SHOW DNA DAMAGE SENSITIVITY SENSITIVITY. THEY HAVE SHOWED GENOME IN INSTABILITY AND OF COURSE IN INCREASED MITT /OOTIC RECOMBINATION RECOMBINATION. WHICH IS -- WHICH HAS LADY -- LED TO THIS -- THE SUGGESTION THAT THIS COMPLEX PLAYS AN IMPORTANT ROLE IN PREVENTING CROSSING OVER DURING THE MITTOT MITTOTIC CELL CYCLE. OKAY, SO WHAT ARE THE ACTIVITIES OF THIS COMPLEX? WELL, AS /I ALREADY ALLUDED TO, B BLMSGS 1 IS A HELOCASE WITH SORT OF BROAD SPEC /TPEIFICITY AGAINST BRANCH DNA MOLECULES. SO JUST LIKES TO BIND TO BRANCH DNA MOLECULES AND TO UNWIND. BY CONTRAST, /TKPWHROERPL AASE 3 AND ITS PARTNER PROTEIN RMI 1 ISN'T REALLY A TOP LIGHT. ARE IT'S A SINGLE STRAND. SO RATHER THAN RE/HRABLAXING DOUBLE STRANDED DNA, WHAT IT ACTUALLY DOES IS IT PASSAGES SINGLE STRANDS TO EACH OTHER BY OPENING ONE SINGLE STRAND AND AALLOWING THE OTHER SINGLE STRAND TO PASS THROUGH. I HAVE TO SAY THAT IN /THIS VENUE IT'S VERY FRUSTRATING BECAUSE I USUALLY USE MY LANDS -- /HAEUPBDZ LOT IN GIVING A TALK. AND THAT'S OBVIOUSLY POSSIBLE. OKAY. SO ONE OF THE ACTIVITIES THAT THE SGS 1 AND FGR HAS THAT IS RELEVANT TO ANTI-CROSS /KWROEOVER ACTIVITY IN, IN FACT, ITS ACTIVITY AGAINST EARLY STRAND IN INVASION INTERMEDIATE AND. WHAT IT HAS BEEN SHOWN TO DO IN VITRO IS BASICALLY TAKE THESE APART HAT HAS BEEN PRIMED BY THE AND IF THERE IS SOME SIN /TYNTHESIS INVADING STRAND, THEN THIS DIS DISPLACED STRAND CAN REALLELE WITH THE EVENED OF THE DOUBLE STRAND BREAK AND WHEN YOU FILL EVERYTHING IN, YOU'VE GOT AN -- A NON-CROSS /KWROEOVER RECOMBINANT. ASSASI A CONSEQUENCE THIS INTER INTERMEDIATE, WHICH OTHERWISE COULD HAVE GONE TON FORM A DOUBLE HOLIDAY JUNCTION /SWR-S IS DIRECTED TOWARDS THE NON- NON-CROSS /KWROEOVER. SO THE QUESTION IS IS THIS RELEVANT TO WHAT WE SEE IN MYOT MYOTIC RECOMBINATION? SO BEFORE I TELL YOU ABOUT THE STUDIES THAT HAR REED AND AR NO DID TO EXAMINE THE ROLES OF THE FUNCTION OF S /T-FPTR DURING MYOTIC RE RECOMBINATION, I WANT TO INTRODUCE YOU TO A COUPLE OF OTHER TOOLS THAT WE USED IN THE SYSTEM. THE FIRST, BECAUSE -- SO AS YOU CAN IMAGINE, WHAT I'VE TOLD YOU ABOUT THIS PROTEIN COMPLEX WOULD INDICATE THAT IT HAS IMPORTANT ROLES IN MYOTIC CELLS. AND IN FACT, MUTANTS IN /TTHIS MUTANT SLACK -- LACKING ANY OF THE THREE MEMBERS OF THIS COMPLEX IN PARTICULAR TOP 3 AND MRI 1 ARE SO UNSTABLE DURING THE MITT /OOTIC CELL CYCLE THAT IT'S DIFFICULT TO GROW UP ENOUGH CELLS TO DO A MYOTIC EXPERIMENT. SO TO AVOID CONTRIBUTIONS OF LESIONS DURING THE MYOTIC CELL CYCLE, WE MAKE STENEXTENSIVE USE OF MY /OOSIS-SPECIFIC OFF ALLELES AND THESE /HRAOELZ RE/PHRAEPLACED THE PROMOTER WITH A PROMOTER THAT IS EXPRESSED DURING THE MYOTIC CELL CYCLE BUT NOT DURING MY /OOSIS. THE SECOND TOOL THAT WE MAKE EX EX/TEPTENSIVE USE OF AND I'LL SHOW YOU IN A MINUTE IS A GRATUITY USELY INDUCIBLE /PROERPBLT CONSTRUCT AND IT USES A GAL ONE PROMOTER WITH A GAL FOUR ACT ACTIVATOR THAT'S INFUSED TO AN ESTROGEN RECEPTER SO THAT IT'S ONLY ACTIVE WHEN YOU ADD EEXTRA EEXTRADYLE TO THE IMMEDIAMEDIUM. SO AS A RESULT, FOR EXAMPLE, IN THE ABSENCE OF CD C 5, WE CAN SEND CELLS THROUGH MY /OOSIS IN THE ABSENCE OF POLLOKINASE AND AS A RESULT, THEY WILL ACCUMULATE NON-CROSSOVERS. THEY WILL ACCUMULATE RE RECOMBINATION INTERMEDIATES BUT THEY WON'T RE/SOSOLVE THEM AND THEN BY ADDING ESTROGEN, WE CAN SIMPLY TURN ON CDC /1K35* DRIVE THE MOLECULE TO A RESOLUTION. SO ONE SUCH EXPERIMENT IS SHOWN IN THE NEXT SLIDE AND I AM GOING TO USE THIS BASICALLY TO SUMMARR -- SUMMARIZE EVERYTHING WE'VE LEARNED IN OUR STUDIES OF STR. SO HERE IS AN EXPERIMENT WHERE WE RUN CELLS THROUGH MY /OOSIS IN THE ABSENCE OF CDC /1K35* TURN DONE AND AS YOU CAN SEE WHAT HAPPENS IS WE ACCUMULATE REBEGIN RECOMBINATION INTERMEDIATES, DOUBLE HOLIDAY JUNCTIONS. WE ACCUMULATE HIGH LEVELS OF NON-CROSS THEOVERS BUT NOT THAT MANY CROSSOVERS. THEN WHEN WE TURN ON CDC 5, THESE JOINT MOLECULES ARE RE RESOLVED AND AS A RESULT, WE SEE AN /EUINCREASE OF CROSSOVERS AND THAT'S SUMMARIZED HERE. WE HAVE DOUBLE STRAND BREAKS, BEING IN THE ABSENCE OF CDC 5 FORMING NON-CROSSOVERS OR DOUBLE HOLIDAY JUNCTIONS. WE TURN ON CDC 5, WE SEE CROSS CROSSOVERS AND WHAT I AM NOT SHOWING YOU IS THIS REACTION IS CAT /HRAOEALYZED BY THE MYOTIC RE/SOFL RESOLVING. IN CONTRAST, WHEN WE LOOK AT A MUTANT, A NUMBER OF THINGS ARE DIFFERENT. FIRST OF ALL /SKWR-RB, IN THE -- IN ADDITION TO DOUBLE HOLIDAY JUNCTION IS WE'RE ACCUMULATE ING A LARGE NUMBER OF AB/ERRANT JOINT MOLECULES THAT HAVE 3 AND EVEN 4 CHROMOSOMES INVOLVED. ADDITION, WE'RE NOT FORMING HER CROSSOVERS OR NON-CROSS NON-CROSSOVERS. SO IT'S ONLY WHEN YOU INDUCE -- WHEN YOU EXPRESS CDC /1K35* TRIGGER JOINT MOLECULE RES RESOLUTION THAT NOW THESE JOINT MOLECULES ARE BEING RESOLVED NOT JUST AS /KROTSOVERS BUT AS CROSS CROSSOVERS AND NON-CROSSOVERS. WHAT I AM NOT GOING TO SHOW YOU HERE TODAY BUT WHAT WE'VE /SHOUPBS THAT THIS RESOLUTION OF THESE AB/ERR STRAND MOLECULES, AS CROSSOVERS AND NON-CROSSOVER! NON-CROSSOVERS, IS CAT /HRAOEALYZED EXCLUSIVELY BY THE MITT /OOTIC RE RE/SOSOLVE AGENT, NOT THE MYOTIC RE RE/SOSOLVE AGENT. AND FINALLY, IN /AA TOP 3 MUTANT WE SEE A SIMILAR PICTURE, AGAIN ABENT MOLECULES, NO CROSSOVERS OR NON-CROSSOVERS UNTIL WE TURN CDC 5 ON. BUT IN ADDITION TO WHAT WE SEE IN SGS ONE, A SUBSTANTIAL FRACTION, BAY THYROID HALF REMAIN UNRESOLVED. SO A JOINT MOLECULE THAT ARE FORMED IN THE ABSENCE OF THE POLY/PHMERASE 3 HAVE A PROBLEM RE RE/SOSOLVING AND WE'LL GET TO WHAT WE THINK IS GOING ON IN A MINUTE MINUTE. SO JUST TO SUMMARIZE WHAT WE'VE LEARNED FROM THESE STUDIES, SGS ONE, TOP 3 AND RMI 1 BASICALLY HAVE 3 ACTIVITIES. ONE, THEY PROMOTE THE EARLY FORMATION OF NON-CROSSOVERS, THE RE/SOSOLVEATES INDEPENDENT FORMATION OF US NON-/KROTSOVERS. SECOND, THEY BLOCK THROUGH THIS OFF-PATH WWAY THAT I DESCRIBED THAT FORMS AB/ERENT STRAND MOLECULES AND USES THE MITT /OOTIC RESOLVING. THIRD, S /T-FPTR MOMENTS -- PROMOTES THE USE OF THIS PATH WAY WHEREBY DOUBLE HOLIDAY JUNCTIONS ARE FORMED IN THE CON CONTEXT OF THE RECOMBINATION MOD MODULE AND ARE RESOLVED EXCLUSIVELY AS CROSSOVERS. SO BASICALLY, TO PUT THIS IN MOLECULAR TERMS, WHAT WE THINK IS /TKPWGOING ON IS THAT SF -- STR ARE ALL INVOLVED IN TAKING APART THE INTERMEDIATES. THE HELOCASE OF SGS ONE IS IN SOME WAYS PUSHING THIS BRANCH AND UNWINDING, BUT AS YOU UNWIND UNWIND, THERE IS A REQUIREMENT FOR STRAND PASSAGE AT /THIS JUNCTION, AND TOP 3 AND RMI ONE ARE /STPOPBL -- RESPONSIBLE FOR THAT STRAND TEST. SO THIS LEADS -- LEAVES WITH US A BIT OF A PARADOX BECAUSE WHAT WE'RE SUGGESTING IS HA THIS ACTIVITY, SEATBELT, THE ACTIVITY OF THIS COMPLEX IS ALL INVOLVED IN TAKING THINGS APART. SO IT'S EASY TO UNDERSTAND HOW, IF YOU TAKE THIS APART, YOU CAN MAKE A NON-CROSS /KWROEOVER. IT'S ALSO EASY TO UNDERSTAND HOW F YOU TAKE SOMETHING APART, YOU CAN PREVENT THE FORMATION OF THESE AB/ERRANT JOINT MOLECULES. BUT HOW AND AN ACTIVITY THAT TAKES THINGS APART BE RESPONSIBLE FOR PROMOTING FLUX THROUGH A PATH WWAY THAT'S ALL ABOUT STABILIZING JOINT MOLL MOLECULES AND PUTTING THEM TOGETHER? AND SO THAT'S THE PARADOX WE'RE LIVING WITH FOR SOME TIME UNTIL I TALKED TO PROTEIN FOLDING COLLEAGUE OF MINE WHO REMINDED ME OF WHAT SHCHAPERONS DO AND PROTEIN SHCHAPERONS DO. AND WHAT HE REMINDED ME WAS SHAP CHAPERONES ACTUALLY DON'T PROMOTE THE FOLD /TPOELG OF THE PROTEINS. WHAT THEY DO IS THEY PROMOTE THE UNFOLDING OF THE PROTEINS. SO THEY RECOGNIZE PARTIALLY UN UNFOLDED INTERMEDIATES AND THEY JUST UNFOLD THEM TOGETHER -- /STKPWEFPBLT SO BY DOING SO, THESE POP /PHRAEPLATE THE ENERGY MINUTEMA THAT IS OCCUPIED BY PARTIALLY UNFOLDED INTERMEDIATE. AND AS A RESULT, PO/PHRAEPLATE THE ENERGY MINUIMUM THAT IS -- THAT IS OCCUPIED BY FULLY FOLDED INTERMEDIATES, WHICH OF COURSE ARE NO LONGER RECOGNIZED BY THE SHCHAPERON. SO BY BEING FULLY FOLDED AND NO LONGER BEING RECOGNIZED BY THE SHCHAPERON THAT THE UNFOLDING ACTIVITY OF A CHAPERONES ARE ABLE TO DIRECT PROTEIN FOLDING INTO THE PROPER AGENT AND WE THINK THE SAME THING IS GOING ON WITH SGS ONE. THE PRIMARYARY ACTIVITY OF STR IS TO TAKE APART RECOMBINATION INTERMEDIATES. AND WHAT WE'RE SUGGESTING IS THAT'S BASICALLY ALL IT DOES. SO WHEN ONE GETS AN EARLY STRAND IN INVASION INTERMEDIATE, CAN -- IT CANNOT -- CAN BE TAKEN APART BY SGS ONE AND RETURN TO THE GROUND STATE. HA WE SUGGEST IS THAT IN FACT DURING THE NORMAL COURSE OF MYOT MYOTIC RECOMBINATION, THERE ARE MULTIPLE CYCLES OF STRAND IN INVASION AND DIS/A /SEASSEMBLY. AND THAT THE ONLY WAY THAT EVENTS CAN ESCAPE THESE CYCLES OF STRAND INVASION AND DIS/ASSEM BLIIS TO FORM A STRUCTURE THAT IS NO LONGER RECOGNIZED BY STR. ONE SUCH STRUCTURE ARE IS A LIN LINEAR NON-CROSS /KWROEOVER PRODUCT. AND BECAUSE IT'S LINEAR, IT WON'T BE RECOGNIZED BY S /T-FPTR. ANOTHER SUCH OUTCOME IS A DOUBLE HOLIDAY JUNCTION THAT HAS BEEN CAPTURED AND PROTECTED IN THE CONTEXT OF THE RECOMBINATION NOD NODULE WHERE THERE ARE SPECIFIC PROTEINS THAT ARE SPECIALIZED TOP STABILIZING THESE INTERMED INTERMEDIATES AND QUITE POSSIBLY TO PREVENTING THE ACTIVITY OF ST STR AS WELL. SO IN THAT WAY, THE DIS/A /SEASSEMBLY OF ACT OF S /P-FPPR ACTING AS /AEA SHAP CHAPERON DRIVES EVENTS EITHER TO THE TWO PRIMARY OUTCOMES OF MYOT MYOTIC RECOMBINATION, TOWARDS NON-CROSSOVERS AND TOWARDS DOUBLE HOLIDAY JUNCTION THAT'S WILL THEN BE RESOLVED AS CROSS CROSSOVERS. SO ONE OF THE PREDICTIONS OF THIS MODEL IS THAT IF THERE ARE REPEATED CYCLES OF INVASION IN DIS/A /SEASSEMBLY, THAT THERE IS A POSSIBILITY FOR PARTNER SWITCHING. SO IF, FOR EXAMPLE, THERE IS FIRST AN /EUINVASION OF A RED CHROMOSOME AND DIS ASEM /SKPWHREU THEN INVASION OF A BLUE CHROMOSOME AND DIS/A /SEPASSEMBLY AND THEN ININVESTIGATION OF A RED AGAIN, FOR EXAMPLE, ONE WOULD EXPECT THAT AMONG THE RE REBEGCOMBINANTS THAT EVENTUALLY EMERGE FROM THIS PROCESS, MOSAIC CONTRIBUTION TO THE REBEGCOMBINANT AND. AND IN FACT, THERE IS SOME EVIDENCE THAT IN FACT THAT HAS HAPPENED -- IN, IN FACT, WHAT'S HAPPENED. SO THIS PAPER FROM THE LAB STUDY STUDIED A FORM OF RECOMBINATION IN THE CELL TO INDUCE REPRESENT REPLICATION. AND WHAT SMITH SHOWED WAS THAT IN THE FIRST TEN KB OF REPRESENT REPLICATION, THERE WERE FREQUENT PARTNER SWITCHES SO THAT THE RE RECOMBINATION EVENTS SWITCHED FROM ONE CHROMOSOME TO ANOTHER CHROMOSOME AS IT WAS BEING REpREPAIRED. SECOND, IN MY /OOSIS, IN STUDIES OF MY /OOSIS USING STRAINS THAT ARE HIGHLY POLYMORPH IC AND THEREFORE CONTAIN A /HROLOT OF POTENTIAL MARKERS AND STRAINS THAT ARE MISMATCHED SO THAT IT CAN'T MISMATCH CORRECT HETERO HETERODUNE PLEX THAT'S FORMED WITH THESE MARKERS, A MANUAL MARTINI SHOWED THAT ABOUT A THIRD OF THE MYOTIC RE/KPWEUPBTS THAT THEY WERE ABLE TO RECOVER AND SEQUENCE CONTAINED COMPLEX PARENTAL CONTRIBUTION. EVENTS THAT CAN ONLY BE EXPLAINED IF THERE WERE PARTNER SWITCHING. SO AGAIN, EVIDENCE IN FAVOR OF THE IDEA THAT IT'S CYCLES OF DIS DIS/A /SEASSEM /SPWHREU RE/EUPINVASION THAT ARE DRIVING THE POPULATION OF THE TWO PREDOMINANT PRODUCT OF MYOTIC RECOMBINATION. AND OF COURSE, THIS STRUCTURE IS COMPLEX THAT IS PROVIDING THE SPEC /TPEIFICITY IN MY /OEOSIS THAT A ALLOWS THE FORMATION OF THESE DOUBLE HOLIDAY JUNCTIONS AND THEREFORE CROSSOVERS AT A HIGH FREQUENCY. IN OTHER WORDS, THIS CYCLE IS BASICALLY LIKE A MITT /OOTIC RE RECOMBINATION CYCLE, EXCEPT THAT WHEN ADDED ON THROUGH SPECIALIZE SPECIALIZED MYOTIC STRUCTURES, THE ABILITY TO BE HIGH LEVELS OF PROCESS. FINALLY, YOU WILL REMEMBER -- SO THAT'S THE FIRST LESSON I THINK WE'VE LEARNED FROM THIS STUDY. FINALLY, YOU WILL REMEMBER FROM THE EARLIER DA/TTA THAT I SHOWED YOU, THAT IN ADDITION TO -- SGS ONE MUTANT APPEARED TO PO PO/PHRAEPOPLATE THIS PATHWAY AND FORM A JOINT MOLECULES, NEVERTHELESS THEY WERE EFFICIENTLY RESOLVED AS CROSSOVERS AND NON-CROSSOVERS. BUT 3 MUTANTS AND I SHOULD SAY R RMI ONE MUTANTS SHOWED A RES RESOLUTION DEFECT. AND SO AT 19, SO WE DON'T KNOW -- WE SUGGEST THAT TAUPE 3 AND M MRI 1 ARE HAVING A ROLE THAT PROMOTES JOINT MOLECULE RE RECOMBINATION INTERMEDIATE RES RESOLUTION /SKWRARBGTS LEAST IN THIS THIRD PATH WWAY OF MYOTIC RE RECOMBINATION. SO TO ACT -- ACCOUNT NEURO, WE SUGGEST THAT IN FACT RE RECOMBINATION INTERMEDIATES THAT ARE FORMED ARE NOT JUST SIMPLE, HOLIDAY JUNCTION CONTAINING STRUCTURES, BUT THAT THEY FREQUENTLY CONTAIN OTHER STRUCTURES, OTHER FORMS OF STRANDS -- STRAND INTERTWINING THAT REQUIRE TAUPE 3 AND MR -- R RMI ONE FOR THEIR RESOLUTION AND I'LL JUST SHOW YOU AN I WILL ILLUSTRATION HERE OF ONE HYPE THETICAL STRUCTURE. THIS IS A DOUBLE HOLIDAY JUNCTION. IT CONTAINS JUNCTION HERE AND HERE BUT IN ADDITION IT CONTAINS IN/TTRASTRAND CAPPING THAT DO NOT -- THAT AREN'T OF THE FORM OF DOUBLE /HOPLD JUNCTION AND IN FACT ARE NOT LINKED BY WHAT'S IN APEARING. IT'S THIS KIND OF A STRUCTURE THAT WE SUGGEST ARE REQUIRED FOR IN ORDER TO /RE/SOFL RESOLVE. OKAY, THAT'S ALL TESTIFY SAY ABOUT S /T-FPTR AND IN THE REMAINING TIME IN THE TALK I'D LIKE TO MOVE /OON TO THE SECOND TOPIC, WHICH IS THE HOW CHROMOSOME ENVIRONMENT INFLUENCES THE BIO BIOCHEMICAL PATH WWAY OF RE RECOMBINATION THAT'S USED DURING MY /OOSIS? SO IF YOU WILL REMEMBER, I TOLD YOU THAT DURING THE EARLY /STPEPZ OF MY OAT -- MY /OOSIS THIS CHROMOSOME AXIS IS LAID DOWN AND THAT DOUBLE STRAND BREAK FORMATION OCCURS IN THE CONTEXT OF THIS AXIS. AND IN FACT, THESE AXIS PROTEINS ARE IMPORTANT -- IMPORTANT FOR EFFICIENT DOUBLE STRAND BREAK FORMATION. IN ADDITION, I TOLD YOU THAT THE LAST STEP OF MYOTIC RE RECOMBINATION, NAMELY, THE RES RESOLUTION OF DOUBLE HOLIDAY JUNCTIONS AS CROSSOVERS ALSO OWE OCCURS IN THE CONTEXT OF THIS ACCESS, IN THE CONTEXT OF THE RE RECOMBINATION MODULE THAT SPECIFICALLY RECRUIT TO THE AXIS AXIS-CONTAINING PARTS OF THE SYNAPSE OF THE COMPLEX. SO REALLY, ALL THE STEPS OF MY MYOTSIC RECOMBINATION FROM START TO FINISH OCCUR IN THE CONTEXT OF THE MYOTIC CHROMOSOME AXIS, WHICH IS MADE UP OF PROTEINS IN ADDITION TO -- SO THIS PRESENTS -- IN SOME WAYS ONE WOULD HAVE TO UNDERSTAND THE ENTIRE POTENTIAL OF RECOMBINATION MECH MECHANISMS THAT ARE PRESENT IN MYOTIC CELLS A BIT OF A QUANDRY BECAUSE THIS AXIS IS NOT UNIFORM UNIFORMLY DISTRIBUTED ALONG THE LENGTH OF THE CHROMOSOME. IN FACT, IT'S ENRICHED IN SOME /SKPWAEUZ -- REASONS AND NOT IN OTHERS. SO HERE IS AN EARLY PICTURE THAT WAS TAKEN IN THE LAB. AND I THINK YOU CAN SEE THAT THERE ARE SOME REGIONS WHERE IT'S HIGHLY ENRICHED AND OTHER REGIONS WHERE IT'S VIRTUALITYLY ABSENT. AND SO AS A RESULT, WHEN WE LOOK AT THE INITIATED MYOTIC RE RECOMBINATION, WE'RE LOOKING ONLY AT THOSE EVENTS THAT ARE OCCURRING IN AXIS-ENRICHED REGIONS. AND NETY, A GRADUATE STUDENT IN THE LAB, WANTED TO ASK WELL IS THAT ALL THAT IT CAN DO? WHAT IF WE WERE TO TAKE -- MAKE A DOUBLE STRAND BREAK NAN AXIS DEPLETED REGION? WHAT WOULD HAPPEN? SO YOU CAN'T DO THAT WITH 411. SO IN ORDER TO DO THIS, HE TOOK ADVANTAGE OF A REALLY VERY INTERESTING PAR /SEASITIC ENDOGLUC ASE SYSTEM CALLED VD /TK-FPD. AND THIS IS SUCH AN INTERESTING STORY TESTIFY SHOW YOU EVEN THOUGH IT'S A LITTLE BIT OFF-TOM OFF-TOMIC. CD E EXISTS THIS IS PROTEIN IN INSERTION IN THE DMA ONE GENE, WHICH IS THE MAJOR VAC EUOLE AND IT'S RESPONSIBLE FOR ACIDIFYING THE VACUOLE AND SO SU WOULD THINK WITH /EUINSERTION IN SUCH AN ESSENTIAL GENE SHOULD BE -- BUT NO. BECAUSE WHAT HAPPENS IS WHEN ONE IS EXPRESSED, THE TRANSCRIPT GOES TO THE SITE. IT'S TRANSLATED AS /AA PROTEIN, BUT THEN DD E EXCISES ITSELF, RE RESTORES THE ININ FACT -- INTACT CODING SEQUENCE AND FORMS THE ENDONUCLEIC. SO THIS ENDONUKE LACE DOESN'T HAVE TO DO ANYTHING DURING THE MYOTIC CELL CYCLE. BUT ONCE CELLS ENTER MY /OOSIS, IT IS TRANSPORTED BACK TO THE NUKE NUCLEUS AND IF THERE IS A VMA ONE SEQUENCE THAT DOES NOT CONTAIN DDE, IT RECOGNIZES THAT SEQUENCE. RUTS IT. IT MAKES A DOUBLE STRAND BREAK AND TAKEN -- THAT BREAK IS RE REPAIRED BY GENE CONVERSION FROM THE CONTAINING SEQUENCE, INTEREST BY PROPAGATING THE D DE SEQUENCE THROUGH THE GENOME AND THIS IS BASICALLY A PAR /SEASITIC ELEMENT THAT PROPAGATES ITSELF. I'M SORRY. SO /TKPAO*RPB TOOK ADVANTAGE OF THIS SYSTEM, AND HE BASICALLY SAID WELL, I KNOW WHAT THE RECOGNITION SEQUENCE OF D DE IS. I CAN PUT IN EITHER AN ACCESS-EN ACCESS-ENRICHED REGION OR ACCESS ACCESS-DEPLETED PRESTIGE AND THEN ASK WHAT ARE THE ACTIVITIES THAT CAT /HRAOELIES DOUBLE STRAND BREAK REPAIR WHERE THE BREAK IS FORMED? AND WHAT I AM GOING TO TELL YOU ABOUT TODAY IN THE REMAINING TIME IS SIMPLY THE CHOICE OF THE JUNCTION RE/SOSOLVE ASASES THAT ARE USED IN /TPORFORMING CROSSOVERS. AND WHETHER THESE ARE THE MYOTIC SO WITHOUT GOING INTO TOO MUCH DETAIL, DAR PON CONSTRUCTED A RE RECOMBINATION REPORTER CONSENT THAT HAS A CUT PLATE ON -- ON ONE COPY AND THE INSERT INSERTED DISK EITHER AT AN AXIS- AXIS-ENRICHED LOT SPOT OR AT AN AXIS-DEPLETED SPOT AT 3. AND SO AN 11-INITIATED RE RECOMBINATION OCCURS ABOUT FIVE TIMES MORE FREQUENTLY HERE AND HERE. BUT WHEN D DE IS DRIVING RE RECOMBINATION UP, IT OCCURS WITH EQUAL FREQUENCY RI-- FREQUENCY AT BOTH. CROSSOVERS. YOU CAN SEE THEY ARE HAPPENING AT ABOUT THE SAME FREQUENCY AT BOTH LOCI. BUT EVEN THOUGH CROSSOVERS AND NON-/KROTSOVERS ARE BEING FORMED AT SIMILAR FREQUENCIES IN THESE TWO LOCI, THEY DIFFER QUITE MARKEDLY WITH REGARDS TO DIS DISSOLVE AASE THAT'S USED IN FORM FORMING CROSSOVERS. SO AT THE HOT /HRO*UBGS, MOST THE CROSSOVERS THAT ARE FORMED ARE BEING FORMED BY THE MYOTIC RESOLVE AASE. SO IN THE ABSENCE OF M /HR-FPL 346789, A RECOMBINANT, /KROTSOVERS ARE RE/TKAOUSD BY ABOUT TWO-THIRDS. IN CONTRAST AT THE LOCUS, CROSS CROSSOVERS ARE VIRTUALLY MLH 3 DEPENDENT. IN OTHER WORDS, THESE CROSSOVERS ARE USING THE YOU MITT /OOTIC RE RE/SOSOLVE ATATE. THESE ARE FORMED BY THE MITTOTIC RE/SOSOLVE ATATE. SO JUST TO SUMMARIZE, BASICALLY THE GENOME WE SEE IS PARTITIONED BY THE AXIS INTO TWO REGIONS. ONE /OF WHICH, WHEN DOUBLE STRAND BREAKS FORM IN THE VICINITY OF THE AXIS, THEY CAN BE CAPTURED BY THE PROTEINS OF THE RE RESOLVED BY THE MYOTIC RE/SOSOLVE AGENT. BY CONTRAST, DOUBLE STRAND BREAKS THAT OCCUR OUTSIDE OF THE CONTEXT OF THE AXIS CAN'T BE CAPTURED BY THESE PROTEINS /POEUPBZ AND THEY ARE HANDLED BY THE MITT /OOTIC RESOLVEATES AND WE WOULD SUGGEST ARE BASICALLY BEING REPAIR AND TREATED AS IF THEY WERE BREAKS /TPHAOEURPLD IN YOU MYOTIC CELL. IN OTHER WORDS, BOTH THE MYOTIC AND MYOTIC RECOMBINATION PATH PATHWAYS CO-EXIST WITHIN THE MYOTIC NUKE LASE AND IT IS THE STRUCTURE OF PROTEI IN THE VICINITY OF A DOUBLE STRAND BREAK THAT DETERMINES WHICH BIO BIOCHEMICAL PATHWAY IS GOING TO BE USED FOR REPAIR. SO IN THE INTEREST OF TIME, I AM GOING SKIP OVER /THE NEXT SET /-OF DA/TTA AND SIMPLY GO TO THE CONCLUSION. SO AS /I TOLD YOU BEFORE, MYOTIC AND MYOTIC RECOMBINATION BIOKEM BIOCHEMISTRY CO-EXISTS IN THE MYOTIC NUCLEUS. GENOME LOCATION DETERMINES WHICH RESOLVEATES IS GOING TO PRODUCE CROSSOVERS AND IN PARTICULAR THESE ARTIFICIAL BREAKS ARE THESE ENDONUKE LASE FORMED DOUBLE STRAND BREAKS THAT OCCUR IN AN AXIS-ENRICHED LOCUS. SO IN THE FUTURE, WE'D LIKE -- WE HAVE A NUMBER OF QUESTIONS WE'D LIKE TO ASK. FIRST OF ALL /SKWR-RB, IS THIS CONCLUSION THAT I TOLD YOU AND SPOKE ABOUT AS IF IT WAS GENERAL, IS IT REALLY GENERAL /AOEIZABLE? TODAY WE'VE LOOKED AT TWO LOCI. AND AT THE TIME RIGHT NOW WE'RE CURRENTLY PUTTING INSERTS IN MANY OTHER AXIS ENRICHED REGIONS TO ASK WHETHER THE SAME DICHOTOMY IN TERMS OF RE/SOSOLVE ASE CHOICE IS MADE THERE. BUT THE ULTIMATE TEST OF THE HYPOTHESIS IS GOING TO BE IF WE CAN TAKE AN AXIS-DEPLETED REGION AND BY RECRUITING ARTIFICIALLY RECRUITING PROTEINS OF THE AXIS TO IT, WE CAN NOW TURN IT INTO A HOT LOCKUS THUSS THE PATH WWAY AND THOSE EXPERIMENTS ARE UNDER WAY. THE WORK I DIDN'T SHOW YOU THE DA/TTA SHOWED THAT IN ADDITION TO THIS LOCAL EFFECT, THIS EFFECT OF LOCAL CHROMOSOME STRUCTURE, THERE IS ALSO A NUCLEUS-WIDE EFFECT IN THAT OTHER DOUBLE STRAND BREAKS ARE ACTUALLY REQUIRED TO ACTIVATE THE USE OF THE MYOTIC RE/SOSOLVE ASASE. NURSE, IF A VVE BREAK IS FORMED. IN AN ACCESS-ENRICHED REGION BUT THERE ARE NO OTHER DOUBLE BREAKS IN THE CELL, IT'S ACTUALLY REPAIRED BY THE MITT /OOTIC PATH WAWAY AND NOT THE MYOTIC PATH WWAY. AND WHETHER THIS IS DUE TO CHANGES IN CHROMOSOME STRUCTURE OR A NUCLEUS-WIDE DNA DAMAGE SIGNAL REMAINS TO BE DETERMINED. AND FINALLY, IN ADDITION TO ASKING WHETHER CHROMOSOME ENVIRONMENT IS IM/PABPACTING OTHER SETS OF MYOTIC RECOMBINATION, IN ADDITION TO RESOLUTION, WE'D LIKE TO EXPLORE THE POSSIBILITY THAT THIS IS NOT A MY /OOSIS- MYOSIS-SPECIFIC FEN /OPHENOMENON, THAT THERE ARE MULTIPLE PATHWAYS FOR DOUBLE STRAND BREAKS, REPAIR EVEN IN MY TOTIC CELLS, AS MANY OF YOU KNOW. AND WE ARE WONDERING WHETHER THE -- THERE ARE FEATURES OF CHROME CHROMOSOME STRUCTURE THAT DETERMINE WHICH REPAIR PATHWAY IS GOING TO BE USED AND THAT THE POSSIBILITY THAT EVEN THE MYOTIC GENOME IS PARTITIONED /WRAR TO ITS PATH WWAY. SO THAT'S ALL I HAVE TO SAY TODAY. AS /I TOLD YOU, MOST OF THE WORK I PRESENTED WAS -- WITH REGARDS TO S /T-FPTR WAS DONE BY AR NODE AND CARR, TWO OUTLINSTANDING POSTDOCS, SUPPORTING WORK FROM LEAH JESS JESSUP, WHO DID SOME OF THE EARLIER WORK WITH REGARD TO THE /STKWRAOEUFPLTD PEOPLE IN THE LAB ALSO SUPPORTED THEM. OUR EARLY WORK IN /THIS REGARD WAS OUTCOME OF COLLABORATIONS WITH SHERMAN AND KIM'S LAB AND I'D ALSO LIKE TO ACKNOWLEDGE THE ONGOING CONTRIBUTIONS OF BOTH NEAL HUNTER'S LAB AND OF WOLF HIGHER AND STEVE COLOWSKII'S LAB AND THE PAPER WAS PUBLISHED IN AN ISSUE OF "MOLECULAR CELL" WITH ACCOMPANYING PAPERS FROM NEAL AND FROM WOLFE, AND I EN ENCOURAGE TO YOU TAKE A LOOK AT THOSE. THE WORK ON VVE WAS DONE ENTIRELY BY DAR PON, A JOINT GRADUATE STUDENT WITH AL STER GOLD /PHA*MAN UNIVERSITY OF SHEFFIELD. AND SO THANKS FOR YOUR ATTENTION ATTENTION. IF YOU WERE PAYING ATTENTION, AND IF WE CAN MAKE IT WORK, I'LL TAKE QUESTIONS. /PHRA [APPLAUSE] >> SO THANK YOU FOR /AA VERY CLEAR AND HE WIELEGANT DESCRIPTION OF A VERY COMPLICATED TOP /KWREUIC. WHAT WE WILL TRY TO DO NOW IS GO TO THE DIFFERENT SITES AND ASK FOR QUESTIONS AT EACH SITE. SO FIRST, DO WE STILL HAVE BALTIMORE ON? DO YOU HAVE ANY QUESTIONS AT BALTIMORE? >> WE DO INDEED HAVE BALTIMORE ON. YES, FIRST OF ALL /SKWR-RB, LET ME CONGRATULATE MIKE FILER TERRIFIC PRESENTATION. THAT WAS REALLY BRILLIANT. I REALLY ENJOYED IT. I HAVE A QUESTION. >> THANK YOU. GREAT. >> SO THERE ARE MANY STEPS BETWEEN AN INITIAL DOUBLE STRAND BREAK AND THE ESTABLISHMENT OF A STRUCTURE THAT HAS TO BE D YOUR -- YOU'RE SUGGESTING THAT THERE HAS TO BE SOMETHING ALONG THAT PATH WWAY, WHICH IS REFLECTIVE OF AN ENVIRONMENT THAT EXISTS PRIOR TO THE DOUBLE STRAND BREAK. THAT HAS SURVIVED THE DOUBLE STRAND BREAK, IN FACT, NOT JUST LOOKING AT A NAKED /SKWRO*EUFPLT HOW DOES THAT WORK? WHERE YOU HAVE A LOCAL SENSE -- CONCENTRATION OF SOMETHING WHICH STAYS AROUND AND STAYS LOCALLY AND SPREADS OVER HERE AND NOT OVER THERE. IS THAT WHAT /YOU ARE THINKING ABOUT? >> YEAH. SO I THINK IT'S PROBABLY OWE OCCURRING BY A NUMBER OF MECH MECHANISMS. ARE UN-ONE /OF WHICH IS, AS YOU SUGGESTED, RECRUITING CERTAIN ACTIVITIES. AND ONE OF THE THINGS WE'RE FLOORING -- EXPLORING IS /THE POSSIBILITY THAT IN FACT STR IN FACT MAY BE RECRUITED, ALTHOUGH THAT STILL A HYPE /THOTHETICAL. BUT IN ADDITION, THERE ARE PROTEIN MODIFICATION ACTIVITIES NAARE RECRUITED TO THE AXIS IN THE VICINITY OF THE DOUBLE STRAND BREAK THAT CERTAINLY CONDITION THE OUTCOME OF RE RECOMBINATION. ONE /OF THOSE, FOR EXAMPLE, IS AS A /HRAOI ALLUDED TO BRIEFLY, THE ME K 1 KINASE, WHICH IS A MY /OOSIS- MYOSIS-SPECIFIC KINASE. IT'S SORT OF AN /AANALOGUE OF CHECK /SWOUPB IT'S A DOWNFAMILY HAVE KINASE. IT'S RECRUITED BY DDR DINE -- KINASE MODIFICATION OF CHROME CHROMOSOMAL PROTEINS N THIS CASE THE CHROMOSOME AXIS AND IT HAS AN ACTIVITY THAT FOPHOSPHORYLATES PROTEINS THAT ARE INVOLVED, FOR EXAMPLE, HOMO/LOGOUS RE RECOMBINATION. SO FOR EXAMPLE, RAS 51 IS A CO- CO-FACTOR IS FOPHOSPHORYLATED BY ME K 1 AND ITS INTER/AOBACTIONS RIN HIBITED. SO THAT'S JUST ANOTHER -- THAT'S JUST ONE EXAMPLE. BUT WE THINK THERE ARE MANY OTHER POST TRANSLATIONAL MODIFICATIONS THAT ARE OCCURRING IN THE VICINITY OF DOUBLE STRAND BREAKS AND ARE MODIFYING RE RECOMBINATIONAL ACTIVITY IN THEIR VICINITY. SO ENS -- FOR EXAMPLE, SEVERAL OF THE COMPLEMENTS OF THE RE RECOMBINATION NODULES ARE KNOWN TO BE /AOUUBIQUITOUS IN LIGASES. WE DON'T HAVE A GOOD IDEA OF WHAT THE TARGETS ARE, BUT THEY HAVE TO BE MODIFYING SOMETHING. SO THAT'S SOMETHING THAT WILL BE INTERESTING. >> YEAH. THE IMPLICATION IS THEY ARE DOING THIS AS -- AT A LOCAL FASHION. NOT BEING TRIGGERED BY THE BRANCH ITSELF OTHERWISE YOU'LL HAVE IT ALL OVER /THE PLACE. THE RESPONSE IS LOCALIZED. >> THAT'S CORRECT. YES. >> OKAY. SO I THINK -- I THINK IT REFLECTS THE SORT O LOCAL NATURE OF THE DNA DAMAGE RESPONSE. VORT DNA DAMAGE RESPONSE LEADS TO DOWNSTREAM OUTPUT OF THE DNA DAMAGE RESPONSE HAS NUCLEASE CELL-WIDE CONSEQUENCES. BUT BEFORE THAT -- AND THIS IS WORK THAT WE AND OTHERS HAVE SHOWN, LOOKING AT H 2 IA.^ FOSS PHOSPHORYLATION IN RESPONSE TO A SINGLE DOUBLE STRAND BREAK^-- YOU GET A LOCALIZED AND MODIFICATION THAT LOCALIZED MODIFICATION RECRUITS PROTEINS AND ACTIVITIES THAT CONDITION THE OUTCOME OF THE REPAIR ITSELF ITSELF. >> OKAY. THANK YOU. >> BALTIMORE. DO WE HAVE A QUESTION FROM RESEARCH TRYING IANGLE PARK IN NORTH CAROLINA? >> YES, WE DO. CAN YOU HEAR US? >> YES, WE CAN. >> HOW ARE YOU? >> I'M GOOD, MIKE. >> GOOD TO SEE YOU.^ ABSOLUTELY TERRIFIC. >> I CAN'T SEE YOU, BUT THAT'S ALL RIGHT. >> WELL, I SEE YOU. SO A QUESTION IS THIS PUTTING A DOUBLE STRAND BREAK AT A SITE WHICH IS DEPLETED BRINGS UP THE QUESTION OF RAND /O*OM DOUBLE STRAND BREAKS, AS MIGHT BE INDUCED BY RADIATION OR SOMETHING LIKE THAT THAT. AND TO WHAT EXTENT OF OTHER KINDS /OF DOUBLE STRAND BREAKS RESPOND? THERE IS A FOLLOWUP OTHER THAN QUESTION. IS THERE ALSO EXCLUSION OF SISTER CHROMATIN RECOMBINATION WITH THE BREAKS THAT OCCUR OUTSIDE THIS AXIS? >> YEAH. OKAY, SO I'LL ANSWER THE SECOND QUESTION FIRST. SO THAT'S THE /STKOEURT -- DIRTY SECRET THAT I DIDN'T TELL YOU ABOUT. AT THE TIME -- SO IT TUGS BOTH SISTER CHROMATINS. AS A RESULT, WE CAN'T REALLY SCORE SISTER CHROMAT ID RECOMBINATION. ALL OF THEM ARE DIRECTED TOWARD THE --. SO WE'RE ACTUALLY WORKING TO TURN DOWN THE ACTIVITY OF D DE BUT FOR THE MOMENT I CAN'T ANSWER THAT QUESTION. WITH REGARDS TO RAND /O*OM DOUBLE STRAND BREAKS, AS YOU, I'M SURE KNOW, AND PROBABLY OOM FOLKS KNOW THAT THERE ARE OTHER ORGANISMS PEOPLE HAVE SHOWN THAT YOU CAN RESCUE THE CHROMOSOME SEGREGATION DEFECT OF 11 MUTANTS BY CAUSING IN/TKOPBENDOGENOUS DOUBLE STRAND BREAKS. BUT THINGS NEVER WORK PERFECTLY. AND WHAT WE THINK -- WHAT WE THINK IS HAPPENING IS THAT WHEN YOU MAKE RAND /O*OM DOUBLE STRAND BREAKS, MOST OF THOSE ARE NOT GOING TO BE EFFECT /KWREUIVE OR THE FRACTION, DEPENDING UPON HOW THE GENOME IS PARTITIONED A GOOD FRACTION OF THOSE ARE NOT GOING BE EFFECT /KWRIVE IN TERMS OF MAKING CROSSOVERS, HOL LOG IN INTERROGATION, ET CETERA. DOES THAT ANSWER THE QUESTION? >> IT KIND OF ANSWERS IT. ARE YOU PREDICTING THAT THEY WOULD NOT BE REBEGINOJGENIC? THEY ARE GOING TO BE PRESUMEABLE PRESUMEABLELY REPAIRED. >> YES. SO I'M SORRY. >> IN WHICH CASE IT COULD BE SISTER -- >> ARE RIGHT. OKAY. SO MY GUESS. WE HAVEN'T DONE THE EXPERIMENTS. WE'RE TRYING TO GET A SYSTEM WHERE WE CAN ACCESS. MY PREDICTION IS THAT BREAKS THAT ARE FORMED IN AXIS COMPLETION VERSIONS ARE PRIME PRIMARILY GOING TO BE REPAIRED BY THE /KRE URS CHROMATIN AND THEY WILL BE IN/KWREFBGT -- IN IN/EFBEFFECT /KWREUFIVE IN DRIVING -- HOMO HOMOLOG. I THINK THAT'S BASICALLY THE PREDICTION. >> OKAY, THANK YOU. >> CAN YOU MUTE IN RESEARCH TRYING IANGLE PARK? DO WE HAVE A QUESTION IN PITS PITTSBURGH? >> HI. WE DON'T HAVE A QUESTION NOW, BUT GREAT TALK, THANK YOU. >> MUTE PLEASE. HOW ABOUT STONE /KWRY BROOK? >> I HAVE NO QUESTIONS, THANK YOU. >> THANK YOU. ANN ARBOR? >> GREAT TALK. NO QUESTIONS FROM US EITHER, THANK YOU. >> THANK YOU. HOW ABOUT ALABAMA? STILL WITH US? OKAY, HOW ABOUT PORTLAND, ORGANIOREGO OREGON? LEXINGTON, KEN /TUTUCKY, STILL ON THE LINE WITH US? >> YES, WE'RE STILL HERE. THIS IS /KPWRAEUR GORDON. I WANT TO THANK MIKE FILER VERY NICE TALK. FOR /AA VERY NICE TALK. I WANT TO GO BACK A LITTLE BIT OF THE SGS ONE INTER/AOBACTION WAS FIRST DISCOVERED. AND THAT WAS TO EXPLAIN -- EXPLAINED BY SGS ONE GENERATE SOMETHING INTERMEDIATES THAT COULD NOT BE RESOLVED. THAT WAS A MITT /OOTIC SITUATION, BUT COULD YOU ELABORATE ON WHAT YOU THINK IS ACTUALLY GOING ON WITH THESE ONE MUTANTS -- I'M SORRY, AND IS THAT NOW YOUR IDEA BASED ON YOUR MYOTIC RESULTS? >> YEAH, SO WHAT I CAN SAY ABOUT THAT IS -- THE FIRST THING IS I DON'T KNOW WHAT THE DEFECT IS IN 3 MUTANTS JUST BY /WAEWAY OF E ELABORATION. TAUPE 3 ARE SLOW-GROWING, AND THEY HAVE REAL CHROMOSOME SEG SEGREGATION DEFECTS. AND THAT CAN BE SUPPRESSED TO A LARGE EXTENT BY KNOCK OUT SGS ONE. IN FACT, THAT'S HOW IT GOT ITS NAME -- SUPPRESSOR -- SLOW GROWTH SUPPRESSOR." IN MY /OOSIS, THAT'S NOT THE CASE. SO THE FEPHENOTYPE I DESCRIBED FOR A TAUPE 3 MUTANT, IN OTHER WORDS WORDS, MAKES AB/ERRANT RE RECOMBINATION INTERMEDIATES AND CAN'T RE/SOSOLVE THEM. IT'S ALSO TRUE FOR SGS ONE TAUPE 3 DOUBLE MUTANT. SO WHEN IT /KUPCOMES TO DOUBLE STRAND BREAK REPAIRS ITSELF, I THINK THE IDEA THAT SGS ONE IS DOING SOMETHING IN THE ABSENCE OUBLE -- TROUBLE, I DON'T OF TAUPE 3 THAT GETS THINGS IN THINK IS CORRECT. SO THEN ONE IS LEFT WITH THE QUESTION OF WELL THEN WHAT IS GOING ON IN THE MYOTIC CELL CYCLE THAT IS NOT HAPPENING IN THE DURING MY /OOSIS, WHERE WE'RE LOOKING AT DOUBLE STRAND BREAK REPAIR? AND I VINYL GLIB ANSWER, WHICH IS THAT THE DEFECT IS NOT BEING PRE-- PROVOKED BY DOUBLE STRAND BREAK. SO THE SUGGESTION IS THAT THINGS THAT ARE GOING WRONG IN THE REPRESELICATION WORK WHERE THERE IS A POSSIBILITY FOR STRAND IN INVASION, STRAND -- THE FORMATION OF TRANSIENT JOINT MOLECULES DON'T HAVE THE STRUCTURE OF THE TYPE WE THINK OF FOR DOUBLE STRAND BREAK RE REPAIR. ARE THE ONES THAT ARE BEING ACT ON BY SGS ONE. TO MAKE MATTERS WORSE IN THE AB ABSENCE DID AND THAT'S THE BEST I CAN DO. >> OKAY. THANKS FOR YOUR TALK. >> GREAT. SO LET'S SEE IF WE CAN ACTUALLY DO THIS. SO UNLESS WE HAVE OTHER BURNING QUESTIONS THAT ANY -- AT ANY OTHER SITE, I JUST WANT /TO THANK EVERYONE. IT LOOKS LIKE WE'VE SURVIVED WITHOUT KEN. THERE WILL NOT BE THROUGHOUT THE SUMMER ANOTHER VIDEOCONFERENCE UNTIL SEPTEMBER 15TH, WHEN WE'LL HEAR FROM THE YOUNG INVESTIGATORS. THANKS, EVERYONE, FOR THEIR PARTICIPATION. /PHRA [APPLAUSE]