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Genetic Study of Checkpoint Defects of the Mus81-1 Mutant in the Fission Yeast Schizosaccharomyces Pombe

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Genetic Study of Checkpoint Defects of the Mus81-1 Mutant in the Fission Yeast Schizosaccharomyces Pombe Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2019 Genetic Study of Checkpoint Defects of the Mus81-1 Mutant in the Fission Yeast Schizosaccharomyces Pombe Darlington Osei Abrefa Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Immunology and Infectious Disease Commons, and the Microbiology Commons Repository Citation Abrefa, Darlington Osei, "Genetic Study of Checkpoint Defects of the Mus81-1 Mutant in the Fission Yeast Schizosaccharomyces Pombe" (2019). Browse all Theses and Dissertations. 2274. https://corescholar.libraries.wright.edu/etd_all/2274 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. GENETIC STUDY OF CHECKPOINT DEFECTS OF THE MUS81-1 MUTANT IN THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science by DARLINGTON OSEI ABREFA B.S., in Molecular Biology &Biotech. University of Cape Coast, Ghana, 2014 2019 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL 11/07/2019 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Darlington Osei Abrefa ENTITLED Genetic Study Of Checkpoint Defects of the Mus81-1 Mutant In the Fission Yeast Schizosaccharomyces Pombe BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ________________________ Yong-jie Xu, M.D., Ph.D. Thesis Director __________________________ Dawn P. Wooley, Ph.D. Program Coordinator, Microbiology and Immunology Committee on Final Examination: _____________________________ Dawn P. Wooley, Ph.D. _____________________________ Michael G. Kemp, Ph. D. _____________________________ Nancy J. Bigley, Ph. D. _____________________________ Barry Milligan, Ph.D. Interim Dean of the Graduate School ABSTRACT Abrefa, Darlington Osei. M.S., Microbiology and Immunology graduate program, Wright State University, 2019. Genetic Study of Checkpoint Defects of the Mus81-1 Mutant in the Fission Yeast Schizosaccharomyces Pombe. In response to various perturbations of DNA replication, the DNA replication checkpoint is activated in eukaryotes to stimulate a cascade of cellular responses that are crucial for maintaining genome stability and cell survival. Defects in the checkpoint pathway result in mutations and genome instability, which is a hallmark for cancers. This study used a genetic approach to identify a mutation in the MMS (methyl methanesulfonate) and UV-sensitive protein Mus81, a DNA repair enzyme that resolves aberrant DNA structures through the homologous recombination pathway. We show that a single missense mutation, identified in fission yeast mus81-1, causes moderate reduction in the phosphorylation levels of the major DNA replication checkpoint proteins Mrc1(Claspin) and Cds1(Chk2) in fission yeast. We also show that the mutation directly affects the DNA repair and the DNA damage checkpoint mediated by Chk1 that causes dramatic cell lethality in mus81-1 mutant upon treatment with the DNA damaging agents: MMS, UV and Bleomycin. III TABLE OF CONTENTS INTRODUCTION ............................................................................................................. 1 The Checkpoint Signaling Pathways .............................................................................. 1 MUS81 ................................................................................................................................ 5 The agents used to activate the DNA Replication Checkpoint ...................................... 6 The fission yeast Schizosaccharomyces pombe ................................................................ 9 HYPOTHESIS AND OBJECTIVES ............................................................................... 9 Hypothesis .......................................................................................................................... 9 Main Objective .................................................................................................................. 9 Specific Objectives ............................................................................................................. 9 MATERIALS AND METHODS .................................................................................... 10 Yeast Strains, Plasmids and Chemicals ......................................................................... 10 Drug Sensitivity Test ....................................................................................................... 10 Immunoprecipitation (IP) ............................................................................................... 11 Western blotting analyses of phosphorylated Mrc1, Cds1 and Chk1 ....................... 11 Flow cytometry ............................................................................................................... 12 Microscopy ...................................................................................................................... 12 IV RESULTS ........................................................................................................................ 14 Drug sensitivity of SN106 mutant ................................................................................. 14 Identification of the mutation and mutated gene in SN106 mutant .......................... 17 Confirmation of the mutated gene; mus81 in the mutant SN106 .............................. 20 DISCUSSION .................................................................................................................. 34 CONCLUSION ............................................................................................................... 36 REFERENCES ............................................................................................................... 37 V LIST OF FIGURES Figure 1: Checkpoint signaling pathway in the fission yeast Schizosaccharomyces ... 2 Figure 2: Role of Mus81 in cell cycle .............................................................................. 7 Figure 3: Crystal structure of human Mus81 complex ................................................. 8 Figure 4: Drug sensitivity of SN106 mutant. ................................................................ 16 Figure 5: Drug sensitivity of SN106 mutant transformed with FPL .......................... 18 Figure 6: Restriction analysis of plasmids using enzyme HindIII .............................. 19 Figure 7: Drug sensitivity of SN106 transformed with WT mus81 and eme1 ........... 20 Figure 8: Identification of the mutation in SN106 mutant by DNA sequencing ....... 21 Figure 9: Mus81 alignment with other eukaryotes....................................................... 22 Figure 10: Nuclease activities of various human Mus81-Eme1 mutants. .................. 23 Figure 11: Mrc1 phosphorylation in the mus81-1 mutant. ......................................... 26 Figure 12: Cds1 phosphorylation in mus81-1 mutant. ................................................ 27 Figure 13: Chk1 phosphorylation in mus81-1 mutant ................................................. 30 Figure 14: FACS analysis of mus81-1 mutant. ............................................................. 31 Figure 15: Double staining analysis of mus81-1 with 0.01%MMS. ............................ 32 Figure 16: Overexpression of cds1 and mrc1 in mus81-1 mutant .............................. 33 VI LIST OF TABLES Table 1 Conserved replication checkpoint proteins in yeast and metazoans .............. 4 Table 2: List of S. pombe strains used in the study ...................................................... 13 Table 3: List of plasmids used in the study ................................................................... 13 Table 4: List of PCR and sequencing primers used in the study ................................ 13 VII ACKNOWLEDGEMENTS I would like to express my profound gratitude to my supervisor Dr. Yong-jie Xu, for his unflinching support that has led to a successful completion of this piece of work. I would also like to thank Dr. Michal Wozniak, Dr. Nafees Ahmad and Dr. Saman Khan in the Xu lab for their remarks and suggestions and again to Dr. Khan for providing the SN106 mutant. I would also like to express my gratitude to the committee members Dr. Nancy Bigley, Dr. Mike Kemp and Dr. Dawn Wooley for their time, advices, suggestions, and constructive criticisms in the completion of this study. Finally I would like to thank my friends and family for making this possible especially my family for the financial support they gave to me. I am particularly thankful to my husband, Samuel Antwi for his immense support and encouragement throughout this study. VIII INTRODUCTION CHECKPOINT SIGNALING PATHWAYS The cell cycle is a highly regulated process in the cell during which DNA is duplicated followed by division of the cell and organelles. The most important stage of the cell cycle, S phase where DNA duplication takes place must be completed with high fidelity to ensure genome stability (Burhans, Carr & Wahl, 2006). Inefficient completion of this phase results in under replication of the genome and unhealthy cells after mitotic division. DNA replication is however constantly under attack by factors of both endogenous and exogenous
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