WELCOME TO THE DNA REPAIR INTEREST GROUP VIDEOCONFERENCE. WE HAVE A VERY BUSY SCHEDULE TODAY. WE HAVE OUR YOUNG INVESTIGATOR SHOWCASE AND WITH DON'T WE START NOW -- WE'LL HAVE THE TALK FIRST AND THE QUESTIONS AT THE END FOR EVERYBODY. SO TAKE IT AWAY PEOPLE AT NIEHS. >> HELLO, EVERYBODY. IT'S MY PLEASURE TO INTRODUCE THE FIRST SPEAKER TODAY MARTA GARBAZC AND SHE GOT HER Ph.D. IN WARSAW POLAND AND THEN JOINED MY LAB IN PURSUING VARIOUS SUBJECTS THAT OUR RESEARCH GROUP IS FOCUSSED ON. ONE OF WHICH SHE WILL TELL YOU ABOUT TODAY. MARTA. >> GOOD AFTERNOON I WOULD LIKE TO THANK THE ORGANIZERS FOR GIVING ME THE OPPORTUNITY TO PRESENT OUR WORK TODAY. I WILL BE TALKING ABOUT THE EVIDENCE THAT CONTRIBUTE TO INITIATION OF DNA REPLICATION. I'M GOING START WITH A BRIEF OVERVIEW OF MODELS OF DIVISION OF THE REPLICATION FORK. THE FORK MODEL OF REPLICATION SHOWED THAT POLYMERASE REFLECT THE GENOME. OVER THE NEXT 20 YEARS NEW REPLICATION FACTORS WERE DISCOVERED AND STUDIES IN VIVO AND IN VITRO SUPPORT LEADING STRANDS REPLICATION OF THE STRANDS. IN 2015 THERE WAS A NEW POTENTIAL MODEL PROPOSED WHERE POLYMERASE DELTA SYNTHESIZES THE STRANDS AND THERE'S A SPECIALIZED STRAW IN ERRORS DURING THE LEADING STRAND SYNTH SYNTHET -- SIN -- SYNTHESIS AND I'M GOING TO FOCUSSED ON UNDAMAGED DNA CHROMOSOMAL REPLICATION AND THE DIFFERENCE IS WHO SYNTHETIZES THE LEADING STRAND. SO FURTHER EXPLORE THE ROLE OF POLYM POLYMERASE DURING THE SYNTHESIS WE COMPARED THE VARIANTS. THE FIRST ONE IS LOOKS AT THE O 26 AND WE COMPARED THAT MUTANT WITH THE WHICH HAS NUCLEUS ONLY AND WE LOOKED AT ONE COPY OF WILD TYPE O2 AND SECOND TOPPY WAS FOR 4 OR 16. WE CONSTRUCTED THE MUTANTS THAT COMMONLY USE THE VARIANTS THAT WE USED IN OUR LABORATORY. WE ANALYZED AND THIS APPROACH ALLOWED US TO LOOK AT COLONIES. SO THE WILD TYPE AND THE 4 LOOKED AT WHAT WAS VISIBLE IN THREE DAYS. WHILE AT THE SAME TIME THE MICROCOLONIES -- AND WE NEED TO WAIT ABOUT 12 DAYS TO BE ABLE TO SEE THE COLONIES. WHILE WORKING WITH THE MUTANT WE ALSO OBSERVED THAT THIS STRAIN ACQUIRES SUPPRESSERS WHICH MANIFESTS AS COLONIES AMONG SMALL COLONIES IN THE SPORE COLONIES. SO TO MINIMIZE CHANCES OF SPECIFIC SUPPRESSERS WE ALWAYS LOOKED AT THE COLONIES FOR O EXPERIMENTS. THE SIZE OF THE SPORE COLONIES SUGGESTING THE LACK OF THE DOMAINS POSES SEVERE GROWTH DEFECTS. ADDITIONALLY WE SAW THE MUTANTS HAVE DOUBLED TIMES AS COMPARED TO O2-4 AND WILD TYPE AND THEY HAVE LARGER CELL SIZES AND THE NUCLEI HAVE IRREGULAR SHAPE AND COMPARED THEM TO THE WILD TYPE FULL LENGTH OF O2 SUBUNITS. TERRIBLY, THE ANALYSIS REVEALED IT PROGRESSED MOLD MORE SLOWLY THROUGH THE CELL CYCLE AS COMPARED TO WILD TYPE AND O2-4 MUTANTS. SO ALL THIS PHENOTYPES SHOW THE CATALYTIC DOMAINS ARE CRITICAL FOR NORMAL YEAST GROWTH. WE LOOKED AT WHO SYNTHESIZED THE LEADING STRANDS AND TO ANSWER THAT QUESTION WE USED MODIFIED VARIANTS OF DNA POLYMERASE ALREADY IN DNA SPECIFIC SIGNATURES THAT WE CAN FURTHER DETECT. SO THERE'S AN ACTIVE SIDE AND BY MODIFYING THE AMINO ACIDS THAT IS ADJACENT SO THE THYROSINE AND LOOKING AT THE POLYMERASE WE LOOKED AT THE ACTIVITY AND ENZYME VARIANTS HAVE SPECIFIC SIGNATURES AND HAVE MORE NUCLEO TIDES AND WE EN OUR STUDIES WE USED NUCLEOTIDES AND THEY INCORPORATED INTO DNA CAN BE SEEN ACROSS THE ENTIRE GENOME. WE USED THE PROCEDURE AND GENOMIC DNA FROM CELLS WITH THE REMOVAL UNDER ALKALINE CONDITIONS WHICH RESULTS IN THE NUCLEOTIDES. IN THIS KAY -- WAY WE GENERATE LIBRARIES OF FRAGMENTS AND USE NEXT GENERATION SEQUENCING. THIS REPRESENTS META-ANALYSIS TO UPSTREAM AND DOWNSTREAM FROM 200 WELL DEFINED REPLICATION ORIGINS INDICATED HERE AS A.C.S. SIMILAR TO OTHER STUDIES IN OUR LABORATO LABORATORY WE OBSERVED ASYMMETRY IN DNA REPLICATION IN STRAINS WITH VARIOUS POLYMERASE ALPHA AND WE OBSERVED LIGAND STRANDS. THE STRANDS ARE LABELLED IN PURPLE ON THE LEFT SIDE AND IN ORANGE ON THE RIGHT SIDE OF THE ORIGIN. IN CASE OF THE EPSINOME IT'S MARKED ON THE LEFT SIDE OF THE ORIGINS AND IN PURPOSE ON THE RIGHT SIDE OF THE ORIGINS. WHEN WE DELETE THE POLYMERASE BOTH STRANDS ARE SYNTHESIZED IN A SIMILAR FASHION WHICH SHOWS THERE'S LEADING AND LAGGING STRANDS ACROSS THE GENOME WHEN CATALYTIC ACTIVITIES ARE ABSENT. BUT THE SYNTHESIS OF BOTH STRANDS BY POLYMERASE DELTA ARE NOT VERY EFFICIENT. YOU REMEMBER THE YEAST WITHOUT THE ACTIVITIES GROW EXTREMELY SLOWLY. SO THE DATA I SHOWED YOU SO FAR ARE IN AGREEMENT WITH WHERE POLYMERASE IS THE LEADING STRAND REPLICATE AND THERE IS A DIVISION OF LABOR AND I WILL SHARE WITH YOU ONE EXAMPLE. SO THEY USED THE DATA TO CALCULATE THE FRACTION USED BY THE POLYMERASE IN RED. THEN HE USED THE INFORMATION TO CALCULATE WHERE A GIVEN POLYMERASE STARTS IN DNA SYNTHESIS AND WHEN WE LOOK CLOSER TO THE LEADING STRAND, WE SEE THE PEAK FOR POLYMERASE ALPHA LOOKS AT THE A.C.S., THEN POLYMERASE DELTA ALMOST TRAILS THE POLYMERASE ALF -- ALPHA AND IT CORRESPONDS TO THE LENGTH OF SYNTHET IS BY THE POLIER ACE -- POLYMERASE ALPHA AND THE NUCLEOTIDE IS THE FRAGMENT LENGTH. IT SUGGESTS THE POLYMERASE DELTA PERFORMS SYNTHESIS WHICH TAKES OVER THE LARGEST STRAND WHICH IS CONSISTENT WITH THE DATA PUBLISHED LAST YEAR. SO BASED ON OUR IN VIVO RESULTS AND FROM VARIOUS GROUPS PUBLISHED DURING THE LAST FEW YEAR WE LOOKED SO THE COMPLEXES BOUNDED TO THE STRANDED DNA AT ORIGINS AND WILL TRANSITION TO SINGLE STRANDED BINDING MODE AND START MOVING IN OPPOSITE DIRECTIONS. THEN POLYMERASE ALPHA SYNTHESIZES THE FIRST WHICH WILL CONTINUE THE SYNTHESIS UNTIL IT COLLIDES AND WILL BIND TO THE PRIMER AND WILL PERFORM THE MAJORITY OF THE LEADING STRAND REPLICATION. SO FAR WE CANNOT EXCLUDE ANOTHER POSSIBILITY THAT EXTENDS TO A PRIMER THAT WILL SYNTHESIZE THAT IS NOT BOUND TO THE REPLICATION FORK AND CAN BE TRANSLATED BY POLYMERASE DELTA BUT WE FAVORED THE FIRST MODEL OVER THE SECOND ONE BECAUSE SO FAR THERE IS NO DIRECT EVIDENCE FOR SUCH EXTENSIVE MIXED TRANSLATION BY POLYMERASE DELTA. AND IT'S LOOKING AT THE DNA AND IT PERFORMS LEADING STRAND REPLICATION IN THE CATALYTIC DOMAINS AND IMPORTANTLY POLYMER AS A DELTA IS INVOLVED IN THE LEADING STRAND SYNTHESIS. SO I WASN'T TALKING ABOUT THAT TODAY BUT WE LOOKED AT THE MUTANT HAS INCREASED MUTATION RATES SO IN THE FUTURE WE WOULD LIKE TO UNDERSTAND WHAT IS THE SOURCE OF THE MUTATIONS INVOLVED. SO FAR OUR DATA SUGGESTS THE POLYMERASE DATA IS RESPONSIBLE FOR PART OF THE MUTOGENESIS AND WOULD LIKE TO UNDERSTAND THE MECHANISMS OF THE EXPRESSION OF THE MUTATION. -- SX -- SUPPRESSION AND I'D LIKE TO ACKNOWLEDGE ALL HERE AND GIVING ME THE OPPORTUNITY TO WORK IN THE GROUP. AND OTHERS. THANK YOU.