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INVESTIGATION OF THE RELATIONSHIP BETWEEN TELOMERE LENGTH AND DIRECT REPEAT RECOMBINATION RATES IN SACCHAROMYCES CEVEVISIAE Thesis Submitted to The Arts and Sciences of the UNIVERSITY OF DAYTON in Partial Fulfillment of the Requirements for The Degree Master of Science in Biology by Michelle Kathleen Fisher UNIVERSITY OF DAYTON Dayton, Ohio August, 2000 1 APPROVED BY: Dr.‘ Jeffrey j>favenhagen Dr. Jayne Robinson Dr. Panagiotis Tsonis ii ABSTRACT INVESTIGATION OF THE RELATIONSHIP BETWEEN TELOMERE LENGTH AND DIRECT REPEAT RECOMBINATION RATES IN SACCHAROMYCES CEREVISIAE Name: Fisher, Michelle, Kathleen University of Dayton, 2000 Advisor: Dr. J. B. Stavenhagen Telomeres function to protect the ends of chromosomes and to allow complete replication of linear DNA molecules. In yeast, the major replication pathway involves telomerase. A second pathway occurs independently of telomerase through recombination. Using a direct repeat recombination assay it was previously shown that Ci^A-direct repeat recombination is specifically repressed near telomeres in wild-type cells (Stavenhagen and Zakian, 1998). This repression is independent of the ability of the telomere to repress transcription of nearby genes, a phenomenon known as telomere position effect. We hypothesize that the repression of telomere direct repeat recombination is under the control of telomere length. In order to further investigate the telomere direct repeat recombination pathway, S. cerevisiae cells with either short or long telomeres were created in strains harboring yeast telomeric direct repeats, recombination rates were measured, and recombination products were analyzed by genomic Southern blots. Short telomere cells were created by an hdflA loss of function mutation. The rate of recombination specifically increased in hdflA cells showing a preference for the RADI recombination pathway. Recombination rates were similar for hdflA cells grown at a permissive and a non-permissive temperature. Cells with long telomeres were created by harboring a high-copy plasmid containing 800 bps of yeast telomeric DNA, double mutations in rifl and rift genes, or a single mutation in the pifl-m2 gene. The long telomere cells had no significant change in the rate of recombination. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. J. B. Stavenhagen, for his time and help towards the work presented in this thesis. Also, thank you to my committee members Dr. Robinson and Dr. Tsonis for their guidance. I would also like to acknowledge other members of the faculty such as Dr. Rowe, Dr. Hoffman, and Dr. Kearns for their words encouragement and support. Thank you also to Cathy Wolf for giving me the opportunity to explore teaching. I would also like to extend my gratitude to several students in the biology department. I will take many of your smiles and words of wisdom with me. A special acknowledgement goes to the members of the Stavenhagen lab. To Jorge, thanks for all your hard work this summer. I hope you continue to enjoy lab work and can take this project to new levels. To Andy, you are always so helpful. Thanks for listening and always lending an extra hand when I most need one. To Michelle #2, you made training my first undergraduate so easy. You are not only an excellent worker, but a valued friend. To Michelle #3, with all your helpfulness you are essentially a member of our lab. Thanks for your patience and computer expertise. Finally to Chris, we made a good team and I will dearly miss you next year. I will be forever thankful for your presence in my life. iv TABLE OF CONTENTS ABSTRACT.........................................................................................................iii ACKNOWLEDGMENTS....................................................................................iv LIST OF FIGURES............................................................................................ vi LIST OF TABLES..............................................................................................vii INTRODUCTION.............................................................................................. 1 CHAPTER I. MATERIALS AND METHODS............................................. 25 Yeast Transformation Strain Construction Recombination Assay Yeast Genomic DNA Preparations Genomic DNA Digest Southern Blot Gel and Transfer Membrane Fixation and Hybridization Southern Blot Detection II. RESULTS.................................................................................... 37 Analysis using Direct Repeat Assay hdflA Mutant Cells have Increased Rates of C1.3A Direct Repeat Recombination hdflA Recombination Rate is not Dependent on a Permissive Temperature Direct Repeat Recombination Increase Occurs via a RADI -Dependent Recombination Pathway In Cells Harboring Mutations that Increase Telomere Length there is a Modest Increase in Direct Repeat Recombination Recombination in hdflA Cells Occurs via an Excision Type Pathway III. SUMMARY AND CONCLUSIONS......................................58 BIBLIOGRAPHY................................................................................................65 v LIST OF FIGURES 1. Telomere replication............................................................................................. 7 2. Telomerase functions............................................................................................. 9 3. Recombination assay for measuring direct-repeat recombination....................21 4. One-step gene disruption method......................................................................... 28 5. Creation of double and triple knockout C1 -3 A strains.....................................29 6. Placement of the interrupted ADH4 and the LYS2 5’ probes............................35 7. Confirmation of hdfl A cells through PCR and Southern blot analysis......... 40 8. Comparison of direct repeat recombination rates for C1.3A direct repeat experimental and the C4A2 direct repeat control cells....................................... 41 9. Rates of recombination for C1.3A experimental and C4A2 control hdflA cells grown at a non-permissive temperature of 37°C...............................................43 10. Rates of recombination for C1.3A direct repeat cells containing mutations in the RADI or RAD52 genes.......................................................................................... 46 11. C1.3A direct repeat recombination rates for cells with increased telomere length........................................................................................................................49 12. BgHI cuts sites for C1.3A hdflA cells.................................................................52 13. Southern analysis of recombinant chromosomes from hdflA C1.3A direct repeat strains...................................................................................................................... 53 14. BgHI cut sites for C4A2 hdflA cells.................................................................... 55 15. Southern analysis of recombinant chromosomes from hdflA C4A2 direct repeat strains......................................................................................................................56 vi LIST OF TABLES 1. Summary of Saccharomyces cerevisiae telomere binding proteins and their affect on the telomere................................................................................................................11 2. Summary of Strains...................................................................................................27 vii INTRODUCTION General Telomere Structure and Function Telomeres are evolutionarily conserved structures located at the physical ends of linear chromosomes. Experiments performed in the 1930’s in Maize and Drosophila first identified telomeres masking the end of the chromosome from cellular components that would otherwise recognize the ends as broken (Muller, 1938; McClintock, 1939; McClintock, 1941). As a protective cap, telomeres function to prevent degradation by endogenous nucleases and to stop the generation of di-centric chromosomes that could interfere in meiosis (McClintock, 1939). In most organisms, telomeres are composed of tandemly arranged simple sequence repeats. Telomere length varies greatly throughout nature ranging from ~35 bp in single celled ciliates to greater than 35 kb in some species of mice (Zakian, 1995). The length of these repetitive elements differs from cell to cell and chromosome to chromosome. In general, telomeres consist of a G-rich repeat that extends in the 5’ to 3’ direction towards the end of the chromosome and terminate with a single stranded 3’ end. The exact composition of these repetitive elements varies from species to species. For example, Tetrahymena has a C4A2/T2G4 repeat, Oxytrichia has C4A4/T4G4 repeat, yeast has a C1.3A/TG1.3 variable repeat, and vertebrates have a T2AG3/C3TA4 repeat (Greider, 1995). The exception to the simple repetitive elements occurs in Drosophila where telomeres were first discovered. Instead, Drosophila have 1 transposable elements which can be lost and added on to chromosome ends (Sheen and Levis, 1994). In addition to their role in stabilizing chromosomes, telomeres allow for the complete replication of the 3’ lagging strand during DNA replication (Watson, 1972). Most organisms are able to complete the replication of their chromosomes using a specialized DNA polymerase (Greider and Blackburn, 1985). To facilitate the complete replication of DNA, the 3’ G-tail is extended
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