PAP1 IS AN EXTRCOPY SUPPRESSOR OF RAD26:4A, A MUTATION THAT INHIBITS THE INTERPHASE MICROTUBULE DAMAGE CHECKPOINT OF S. POMBE By SHIVANGI PALIWAL B.Sc. Holkar Science College, Indore, India 2007 A thesis submitted to the Graduate Faculty of the University of Colorado Colorado Springs In partial fulfillment of the requirements for the degree of Master of Sciences Department of Biology 2017 Copyright by Shivangi Paliwal 2017 All Rights Reserved This thesis for the Master of Sciences degree by Shivangi Paliwal has been approved for the Department of Biology by Thomas Wolkow, Chair Cheryl Doughty Lisa Hines Date-12/05/2017 ii Paliwal, Shivangi (M.Sc., Biology). Pap1 is an extracopy suppressor of rad26:4A, a mutation that inhibits the interphase microtubule damage checkpoint of S. pombe Thesis directed by Associate Professor Dr. Thomas D. Wolkow ABSTRACT The highly conserved PIKK family protein Rad26 of Saccharomyces pombe is a crucial component of cell cycle checkpoint and the DNA-damage response pathway. The Rad26 is homologous to ATRIP, a human DNA damage repair protein. Recent researches indicate an additional role of Rad26 ATRIP in microtubule damage response that is genetically distinct and independent of the DNA damage response. The aim of this research was to understand this pathway of Rad26 by identifying an extragenic suppressor protein that interacts with Rad26 during microtubule damage response on the treatment of cells with microtubule poison like MBC. The UV mutagenized S.pombe mut2A cells were transformed with genomic library, and the transformants were replica plated on to MBC selective media to identify the plasmid that confers MBC resistance. The plasmid was isolated and sequenced to identify the genes present in the plasmid. Additional transformations with individual clones of genes found in the plasmid resulted in MBC resistance in cells that were transformed with pap1 gene. Sequencing of the pap1 from mut2A revealed an absence of point mutation that leads us to conclude that expression of extra copies of pap1 resulted in MBC resistance instead of the interaction between Rad26 and Mut2A. Pap1 dependent upregulation of the efflux pumps is the hypothesized mechanism for multidrug resistance in S.pombe and a possible mechanism for MBC resistance as well. The identity of the Rad26 interacting protein still requires further research outlined in this thesis. iii ACKNOWLEDGEMENT I would like to express my sincere gratitude to Dr. Thomas Wolkow, for his guidance, expertise and for providing an excellent and flexible research atmosphere throughout the completion of this research work. I would like to thank my committee members Dr. Cheryl Doughty and Dr. Lisa Hines for their time and valuable inputs on the thesis. I would like to thank Dr. Sandra Berry-Lowe for her availability and guidance through the graduate program. Finally, I would like to thank my family for their best wishes and my husband Anand Paliwal for his constant support and encouragement. iv TABLE OF CONTENTS CHAPTER I. INTRODUCTION………………………………………………………………...1 II. REVIEW OF THE LITERATURE……………………………………………....7 The checkpoint………………………………………………………………....7 Rad3 and Rad26………………………………………………………………..8 ATR/ATRIP………………………………………………………………......10 Centrosomes, spindle pole body, and Microtubules…………………………..11 Seckel syndrome………………………………………………………………13 MBC…………………………………………………………………………..14 III. EXPERIMENTAL PROCEDURE………………………………….................16 IV. RESULTS……………………………………………………………...............28 V. DISCUSSION…………………………………………………………………..34 VI. CONCLUSION…………………………………………………………………38 References……………………………………………………………………………39 Appendix……………………………………………………………………………..44 v LIST OF FIGURES FIGURE 1.1 DNA damage response………………………………………………………….....3 1.2 Extragenic suppression………………………………………………………….....6 3.1 Gel image of pTNF2 amplification ………………………………….…..............17 3.2 Transformation of mut2a…………………………………………………………19 3.3 Complementation procedure……………….....………………………………….19 3.4 Gel image of transformed mut2a genomic DNA………………………………...21 3.5 Gel image confirming DNA integrity………….…………………………...........26 3.6 Primers for pap1 sequencing……………………………………………..............27 4.1 Transformed mut2a replica plating…………….………………………………...28 4.2 Replica plate highlighting viable colony ………………………………………...28 4.3 Plasmid complementation……………………...………………………………...29 4.4 Restriction digestion of plasmid………………………………………………….30 4.5 Restriction digestion of plasmid conferring MBC resistance…………................31 4.6 Vector with estimated insert size …………………………………………..……31 4.7 Alignment results………………………………..……………………………….32 4.8 Replica plating results with pap1, hrp1, and sequenced library plasmid…….…..33 vi LIST OF TABLES TABLE 2.1 G2 DNA Damage check point genes……………………………………………..8 vii CHAPTER I INTRODUCTION Genomic integrity and fidelity of cell division are carefully assessed by the DNA Damage response (DDR) pathways known as cell cycle checkpoints. In response to DNA lesions, inadequate growth or adverse environment these pathways delay or arrest the cell cycle progression. These regulatory mechanisms help ensure that DNA integrity is preserved from G1 to S phase and from G2 to M phase of the cell cycle. The replication checkpoint and G2/M checkpoint do this by suspending cell division or triggering cell death depending on the degree and ability of DNA damage repair. These response pathways are complex processes that coordinate among various other pathways. For example, the replication checkpoint (S-phase) suspends progression of cell cycle and coordinates it with DNA repair pathway and recommencement of DNA replication. The G2/M checkpoint monitor and coordinate segregation of sister chromatids with cytokinesis. These cell cycle checkpoints consist of fundamental components of signal transduction pathways called the sensors, transducers, and effectors. A typical signal transduction sensor detects the signal and relays the presence of the signal to transducers that amplify the signal and activate or inhibit effectors. The cell cycle checkpoint sensors can detect single strand overhangs, double-stranded breaks, and abnormal chromatic structure. In S. pombe six `rad' genes (rad1+, rad3, rad9, rad17, rad26, and Hus1) are required for the S and G2/M checkpoints in response to DNA damage. Studies have shown that Rad9-Rad1-Hus1 (9-1-1) complex form a PCNA- like clamp which is loaded on to damaged DNA by Rad17 (Replication factor C subunit) also acts as a sensor and detects DNA damage. Rad3 is a Phosphoinositidiol- 3 kinase-related kinase (PIKK) family member that is also a sensor of cell cycle 1 integrity checkpoints. Studies have shown that the Rad3 and its regulatory subunit Rad26 physically interact like their respective human homologs ATR and ATRIP. In fission yeast the Rad3 interacts with Rad26 and together with the Rad17 and the 9-1-1 complex, detect stress and activate Rad3/Rad26 kinase activity. The Rad3/Rad26 complex (sensors) then phosphorylates and activates evolutionarily conserved transducer Checkpoint kinases (CHK) Chk1 or Cdsl. In the successive step, these transducers, in turn, either inhibit cyclin-dependent kinases (serine/threonine protein kinases) by inhibiting Cdc25 phosphatase effector, or activate Wee1 kinase effectors. This phosphorylation of cdc25 results in its confinement in the cytoplasm by interaction with S.pombe Rad24. Inactive cdc25 cannot dephosphorylate cdc2, simultaneously the dephosphorylation of Wee1 stabilizes itself and prevents its degradation which again phosphorylates cdc2 and blocks it. Inactivation of cdc2 is essential because cdc2 in coordination with other downstream cyclins initiates mitosis. Overall mechanism results in a delay of mitosis that allows extra time for the damage or stress repair pathways to occur before re-entry into the cell cycle. In humans, homologs of these S. pombe genes exist, and their protein products operate in similar fashion (Figure 1). RAD3 and RAD26 homologs Ataxia talengastecia RAD3 related (ATR) and Ataxia talengastecia RAD3 related interacting protein (ATRIP) are also the key proteins of checkpoint signaling in human that activate downstream signaling to inhibit cyclin-dependent kinase activity. 2 a. DNA Stress S.pombe DNA Stress human sensors Rad3/Rad9 or 9-1-1 complex ATR/ATRIP or ATM Checkpoint kinases Transducer Checkpoint kinases s Effectors Cdc25 or wee1 Cdc25A Cdc2 Cdc2 S phase arrest b. G2/M DNA damage ATR ATRIP Rad3 / Rad26 or ATM CHK s CdK s G2/M arrest DNA repair P53(apoptosis) Figure 1.1. DNA damage response a. DNA Damage response in S phase. b. G2/M response In fission yeast, Rad3ATR and Rad26ATRIP are required to overcome interphase microtubule perturbation induced by MT poisons like MBC and TBZ. Baschal et al. (2006) in their study with Rad26 deletion cells, showed the requirement of Rad26 for microtubule-dependent processes including morphogenesis (cell shape) and chromatid separation. They observed accumulation of florescent tagged Rad26 in cytoplasm, especially after MT poisoning. This observation indicated its additional role in a signal transduction pathways associated with damaged microtubules. Microtubules are highly organized forming cytoskeleton which is crucial for cellular morphology, cellular transport, accurate chromatid segregation and cell division. Therefore, abnormal cellular morphology and mini-chromosome loss in Rad26 deficient cells further suggest its additional role. 3 In a follow-up study, Herring et al. (2010) showed that the Rad26-dependent interphase microtubule-damage checkpoint