Pap1 Is an Extrcopy Suppressor of Rad26:4A, a Mutation That Inhibits the Interphase Microtubule Damage Checkpoint of S

PAP1 IS AN EXTRCOPY SUPPRESSOR OF RAD26:4A, A MUTATION THAT INHIBITS THE INTERPHASE MICROTUBULE DAMAGE CHECKPOINT OF S. POMBE

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

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

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.

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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.

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

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

LIST OF TABLES

TABLE

2.1 G2 DNA Damage check point genes……………………………………………..8

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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.

a. DNA Stress S.pombe DNA Stress human

sensors Rad3/Rad9 or 9-1-1 complex ATR/ATRIP or ATM

Transducer Checkpoint kinases Checkpoint kinases s Effectors Cdc25 or wee1 Cdc25A

Cdc2 Cdc2 S phase arrest

b. G2/M DNA damage Rad3 ATR/ Rad26ATRIP 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 delays spindle pole body (fungal

centrosomes) separation and other early mitotic entry events upon interphase

treatment with MT poison. The C terminus of the Rad26 has a conserved nuclear

export sequence (NES). They mutated four hydrophobic residues of the NES to

alanine (rad26:4A allele) which caused the Rad26-GFP signal to disappear from the

cytoplasm. In turn, cells lose this checkpoint response to interphase microtubule

damage and enter mitosis. They also lose chromosomes, viability, and their

cylindrical shape whereas the DNA damage response in the rad26:4A mutated cells

remain functional. These results demonstrate two genetically distinct functions of

Rad26; one responds to DNA damage and the other to microtubule damage that are

mutually independent.

Following the publication of both reports discussed above (Baschal et al. (2006),

and Herring et al. (2010), Graml et al. (2014) corroborated the relationship between cell cycle checkpoint genes and microtubule-dependent processes including cellular morphology, the organization of microtubule and cell cycle. By using microscopy, gene analysis, and bioinformatics tools, they found that most genes that regulate cell shape, microtubules, and cell cycle progression function independently. Interestingly, they also unraveled a link between DNA damage response genes and deregulation of microtubules and microtubule function. The cells with the absence of DNA repair factors Rad55 and Mre11 had abnormally elongated microtubules (more stable) in interphase whereas cells lacking checkpoint protein Rad3 and Tel1 had microtubules like wild types. These results indicate that impaired DNA damage repair brings about stabilization and lengthening of microtubules with the increase in cell length associated with cell cycle arrest. This relationship between DDR and microtubules

elongation is also found in human. These findings indicate a possible connection between cell cycle arrest following DNA damage and cell elongation and cytoskeleton maintenance. These results are of interest to us because it suggests a connection between DDR and proper microtubule maintenance.

To better understand the Rad26-dependent interphase microtubule damage pathway, high school science teacher Bob Wheeler UV mutagenized the rad26:4A strain to identify an extragenic suppressor that allowed rad26:4A cells to grow on

MBC (microtubule poison). He identified one that he named mut2a. When outcrossed from the rad26:4a background, the mutation conferred sensitivity to MBC on its own.

This observation indicates that possibly these two proteins interact with each other.

The focus of this research was to identify and sequence this gene. The knowledge of this gene/protein will lead to better understanding of the exact role of Rad26 in the microtubule damage response pathway.

Identification of the extragenic suppressors is a powerful tool for identifying proteins interactions. Extragenic suppression occurs when a mutation occurring in a different gene complements the original mutation. It is one of the classic methods to identify interacting proteins. For example, if the initial missense mutation results in a change in conformation of the protein and renders pathway non- functional, a complementing alteration in interacting protein conformation due to extragenic suppression will restore interaction and pathway functionality. On the contrary, a deletion mutation can be overcome by bypass suppression that makes up for the requirement of the gene altogether and does not indicate direct protein/protein interaction. A high-copy suppression occurs by expression of extra copies of a gene somewhere downstream in the signal transduction pathway that activates a pathway irrespective of a defect in an upstream pathway component. The protein encoded by

the mut2a gene is an extragenic suppressor of the rad26::4a mutation because the

double mutant is no longer MBC sensitive, while the single mutants are MBC

sensitive. Thus, the mut2a mutation produces a compensatory mutation in a protein

that interacts with Rad26 and acts as an extragenic suppressor of MBC sensitivity.

Sequencing of this gene will potentially identify a protein product that can be further

utilized in the understanding of additional proteins that physically interact with Rad26

or are involved in this pathway and help it respond to MBC damage.

Wild type proteins Mutation affecting suppression restores with functional interaction interaction and interaction (pathway blocked) pathway

Figure 1.2. Extra genic suppression. General mechanism of extragenic suppression.

The Rad26 human homolog ATRIP mutation has been implicated in causing

Seckel Syndrome (SS) characterized by growth retardation, microcephaly, and DNA

damage response defects. Interestingly, mutations in genes encoding centrosome

proteins also cause Seckel Syndrome without affecting DNA damage response

pathways. Evidently, ATRIP also localizes to centrosomes in response to DNA

damage and control microtubule assembly. The localization of ATRIP, RAD26

homolog at centrosomes is a potential indication of Rad26 and spindle pole body

interaction. Identification of the protein that interacts with Rad26ATRIP in the pathway that involves spb and maintaining microtubule assembly might lead to better understanding of Seckel syndrome.

CHAPTER II

LITERATURE REVIEW

The Checkpoint

The cell cycle checkpoint is the pathway that detects and responds to the defects

or errors in the cell cycle. S.pombe has three specific cell cycle checkpoints- 1. G1-S checkpoint monitors for the existence of necessary conditions required for cell division. 2. The G2-M checkpoint prevents the onset of mitosis when DNA replication/synthesis is erroneous.3. The spindle assembly checkpoint monitors the attachment of kinetochores to the spindle assembly and arrests mitosis in case of improper chromosome attachment. The checkpoint “rad genes” is the universal class that is crucial to maintaining the fidelity of cell division (Carr AM, 1995).

Two classes of highly conserved protein kinases are the important upstream components of checkpoint with a phosphatidylinositol 3-kinases (PI3Ks) related domain and a DNA dependent protein kinase domain. The first one that responds to

UV and ionizing radiation are the structural and functional homologs Rad3 and Mec1 of the yeasts, and ATR (ATM and Rad3 related) in mammalian cells. The second

ATM protein (Ataxia Telangiectasia Mutated) respond to damages caused by ionizing radiation with some crosstalk between ATR and ATM. (O’Connell, 2000)

In fission yeast, Rad3 (ATR) and Rad26 (ATRIP) are key components of signaling pathway and exist as a complex. The Rad26 is the regulatory subunit of

Rad3 and is also required for Rad3 expression. Phosphorylation of Rad26 is the first biochemical step of the Rad3 function. (Wolkow, 2002)

Table. 2.1 G2 DNA DAMAGE CHECKPOINT GENES Fission Budding Functional class Humans Comment yeast yeast

BRCT proteins Rhp9 RAD9 BRCA1 Cut5 DPB11

Checkpoint Rad17 Rad24 Rad17 RFC homology Loading complex RFC3 RFC2-5 RFC RFC subunits (CLC)

Chk1 Protein kinase, DIP Effector kinases Chk1 Rad53 Rad17

Cds1 Rad9 Hus1

ATR Rad3 Mec1 Protein kinase ATM Tel1 PI3K-like complexes Tel1 Protein kinase Rad26 Rad3 substrate, DIP Checkpoint Sliding Rad1 Mec3 Ddc1 5′→3′ exonuclease clamp (CSC) Rad9 Chk1 Rad17 DIP (PCNA-like) Hus1 Cds1/Chk2

1. Abbreviations: DIP- DNA damage-induced phosphorylation; PI3K- phosphatidylinositol 3-kinase, Chk- checkpoint kinase O’Connell, (2000) The G2 phase DNA damage check point

Rad3-Rad26

In S. pombe, Rad3 is essential for initiation of all DNA checkpoints but not

required for the viability of cells. Each of the ‘checkpoint rad’ null mutants have

defective DNA-integrity checkpoints. However, rad3 deletion and rad26 deletion are

non-viable with null mutations of rqh1encoding the RecQ-like helicase indicating of

the function during replication. Also, neither rad3 deletion nor rad26 deletion cell fail

to activate Cds1 during S-phase arrest. These observations point to dependent

relationship between Rad3 and Rad26. (Edwards et al.1999). Checkpoint signals alter

Rad3 kinase activity which then activate checkpoint kinases Cds1 and Chk1 by

phosphorylation, eventually delaying entry into mitosis. The dependence of the kinase

activity of Rad3 on Rad26 indicates that Rad3 is the catalytic subunit and Rad26 acts

as a regulatory subunit of the Rad3/Rad26 complex. The Rad3 and Rad26 are important components in sensing and activation of DNA dependent stress pathway, but only specific DNA perturbations result in Rad26 alterations by Rad3. (Wolkow et al. 2002)

Rad26 is a 70 kDa protein with a C- terminal coiled-coil region and basic residues like a nuclear localization signal. (Al-Khodairy et al.1994) Many other functionally conserved proteins with sequence like Rad26 have also been found like the ATRIP in human (Cortez, 2001), LCD1 in S. cerevisiae (Paciotti, 2000) and

UVSD in A. nidulans (De Souza,1999)

The G2 phase DNA damage Checkpoint is dependent on Rad26 in fission yeast

(al-Khodairy, 1994). Rad26, the homolog of human ATRIP is also associated with

checkpoint response induced by microtubule damage. Besides arresting the G2 phase,

delaying spindle pole body separation it also delays DNA replication, on the treatment

of cells with microtubule-targeting drugs. The C-terminal domain of Rad26 has a

Nuclear Export Signal (NES) that is essential for translocation of Rad26 and this NES

dependent localization in not required during checkpoint response. Rad26 localization

to cytoplasm is also essential for microtubule damage response. Actively dividing

cells are unable to arrest mitosis when treated with microtubule poisons if Rad26

remains confined to the nucleus resulting in abnormal cellular morphology and mini-

chromosome loss. (Herring at al. 2010) Budding yeast Ddc2, is functional homolog to

S. pombe Rad26 and is essential for DNA damage checkpoints and hinders spindle

elongation during stalled replication of DNA. The budding yeast Ddc2 and Mec1

interact with each other and Mec1 is phosphorylated during regular cell cycle as well

as DNA damage response just as Rad3 and Rad26 in fission yeast. Evidently, Ddc2–

Mec1 complex respond to DNA perturbations independently of the other checkpoint proteins. This Ddc2 and Mec1 interaction, corroborate kinase–substrate relationship of checkpoint proteins like Rad3 and Rad26. (Paciotti et al. 2000).

As the Rad26-GFP localization in hus1Δ, rad1Δ, rad9Δ, rad17Δ, and rad3Δ backgrounds occur in normal manner it is believed that Rad26 can begin initial steps of the DNA damage checkpoint without these checkpoint proteins, importantly, Rad3.

(Wolkow et al. 2003). Moreover, only Rad3/Rad26 are essential for rescuing cells from microtubule poisons as evident from hus1Δ and rad17Δ cells exhibiting normal

growth on MBC, a microtubule-destabilizing drug. (Herring et al., 2006).

ATR / ATRIP

Cortez et al. identified an ATR-interacting protein (ATRIP) in human that is

phosphorylated by ATR in similar manner as Rad3 and Rad26. It also regulates

expression of ATR, and functions as an important factor of DNA damage checkpoint

pathway. ATR /ATRIP localize to the nucleus after genotoxic stress. Cells deficient in

ATR lack both the ATR and ATRIP expression, and have defective DNA damage

checkpoint responses, leading to apoptosis. ATRIP specifically degraded by siRNA

cause the loss of expression of both ATRIP and ATR and DNA damage response

mechanism proving that ATRIP and ATR have interacting role in cell cycle

checkpoint response. (Cortez et al. 2001). The ssDNA binding protein complex

Replication Protein A (RPA) occupy central role in maintenance of genomic

machinery, and replication errors and genotoxic stresses are sensed through detection

of continuous RPA bound DNA by ATR and ATRIP. (Zou et al. 2003). Breast-

ovarian cancer susceptibility 1 (BRCA1), another checkpoint protein interacts with

ATRIP through Ser (239), a residue that gets phosphorylated. Substitution of Ser(239)

residue of ATRIP with Alanine causes G2-M checkpoint defect suggesting that

physical interaction between both is a necessary aspect of the function of ATRIP.

(Venere et al. 2007). Involvement of ATRIP in other pathways outside nucleus has

also been shown by Zhang et al. (2007) in their experiments with He La cells where

ATRIP localization was observed in centrosomes. The ATRIP deficiency has been

associated with various disease phenotypes including growth retardation,

microcephaly, as observed in Seckel syndrome.

Centrosomes, spindle pole body and Microtubules

Centrosomes are small, membrane-free organelles, with roles in the control of

microtubule dynamics in the cell. A typical centrosome consists of two centrioles, that

are formed by arrangement of nine sets of triplet microtubules symmetrically around a

central axis.

Centrosomes are the main microtubule-organizing centers (MTOCs) of animal

cells. Each centrosome is made up of centrioles embedded in a pericentriolar matrix

(PCM) of proteins. The PCM comprises γ-tubulin ring complexes required for microtubule (MT) nucleation, and few other proteins with coiled-coil domains.

(Moritz et al. 2001). Since yeast lack MT organizing centrosomes, spindle pole bodies

(SPBs) function as microtubule organizing centers in yeast. SPBs are layered, and associate with the nuclear envelope, and therefore structurally distinct from centrosomes. However, both are functionally equivalent because of the use of a

similar γ-tubulin-based mechanism for nucleation of MTs. Centrosomes and SPBs both govern MT-dependent processes, like intracellular organelle distribution and cellular transport, cell shape, spindle formation and cell division. Moreover, mutations in genes encoding proteins of centriole and centrosome have been linked to human disease, like ciliopathies and cerebral diseases. As a result, interest in the studies of structure and function of these organelles have significantly increased.

(Christian et al. 2014). The microtubule network formed during interphase is not only required for cell polarity and intracellular transport but dynamic organization of MTs into spindle assembly is also responsible for proper chromosome segregation.

(Fourest-Lieuvin et al. 2006). Microtubule dynamics that is the continuous alterations in their length is dependent on tubulin and its ability to hydrolyze GTP (Mitchison and Kirschner, 1984) and many of other associated MT effectors. The cell division cycle protein Cdk1 associated with cyclin B is essential for microtubule morphogenesis. Cdk1 phosphorylates MT motors that are implicated in various steps of mitosis including the phosphorylation of Cdk1 itself and regulate localization of microtubules into spindle assembly. (Mishima et al.2004). Studies with mouse embryonic fibroblast cells (MEFs) with mutant Brca1 gene have functional G1–S checkpoint but have impaired G2–M checkpoint and exhibit amplification of functional centrosomes, resulting in the formation of multiple spindle poles. These abnormalities give rise to unequal separation of sister chromatids, defective nuclear division, and aneuploidy signifying an association between checkpoint signaling and microtubule function. (X Xu et al.1999). Various factors involved in the DDR pathway are also detected at centrosomes, including the ATM, ATR, CHK1 and

CHK2 kinases. Regular centrosome organization is also strongly affected by treatment with DNA-damaging agents. (Mullee et al. 2016). DNA damage induced

amplification of centrosomes requires CHK1 and CDK activity. (Bourke et

al. 2010,2007). These findings further illustrate a linkage between the DDR, the cell

cycle machinery, and the centrosome.

Seckel syndrome (SS)

Seckel syndrome, an autosomal recessive disease in human is characterized by growth and mental retardation, small head, and peculiar facial features. The mutation in ataxia telangiectasia and Rad3 related protein (ATR) gene located on chromosome

3q22.1-q24 result in Seckel syndrome. There are 8 different subtypes of SS based on specific gene alteration. CDK5RAP2 gene expression product is a centrosomal protein that is essential for spindle formation and is also responsible for causing SS.

Mutation in CDK5RAP2 results in erroneous mitosis and defective spindle assembly.

(Yigit et al. 2015) Mutations in pericentrin (PCNT) gene leads to loss of matrix from the centrosome that is required for microtubule assembly. Mutations also result in abnormalities in the attachment of structural and regulatory proteins and cause Seckel syndrome. Patients with PCNT (PCNT-Seckel) Seckel syndrome have defective checkpoint signaling (dependent on ATR function), indicate a link between centrosomal structural protein with DNA damage signaling. These findings also indicate that other microcephaly genes involved in either DDR or centrosomal function may be involved in developmental pathways that regulate human body and brain size. (Griffith et al. 2008).

MBC (methyl 2-benzimidazolecarbam) - MT poison

MBC (fungicide) impacts mitosis and cell division, by inhibiting the

polymerization of tubulin to form microtubules and suppressing their dynamic

instability. It induces a conformational change in the tubulin itself hindering

modification of its cysteine residues. The cysteine residues are vital for assembly of

tubulin monomers into microtubule. (Gupta et al. 2004). MBC inhibits proliferation of

cells and arrests mitosis by preventing microtubule dynamics. It induces mitotic

spindle defects and lessens the metaphase centromere space of sister chromatids,

indicating a decrease of tension at kinetochores, initiating cell cycle checkpoints and leading to metaphase arrest and ultimately apoptosis. (Rathinasamy et al. 2006)

Microtubule dynamics are carefully regulated during the cell division cycle by various cellular regulators. Many antitumor drugs and natural compounds also alter the polymerization dynamics of MTs, arresting mitosis, and consequently, inducing cell death by apoptosis. Some of these drugs inhibit microtubule polymerization at high concentrations while some compounds stimulate MT polymerization and stabilize microtubules at high concentrations. Interestingly, at lower levels, all drugs have a common mechanism of action; they suppress the MT dynamics without affecting the mass of microtubules in the cell. Binding of these drugs occur at different sites on tubulin and MT, and they also differ in affecting the microtubule dynamics. However, they all block mitosis at the transition of metaphase/anaphase and induce apoptosis. (Jordan MA, 2008). Benomyl (MBC) inhibits the polymerization of brain tubulin into microtubules and actively suppresses the dynamic instability of brain microtubules without affecting the other parameters. In the studies, it also inhibited proliferation of the He la cells and blocked mitotic spindle function by disturbing microtubule and chromosome organization. Because of its

effect on microtubules and mitosis combined with low toxicity it might be

incorporated as an effective component in oncology treatments. (Gupta et al. 2004).

MBC also disturb microtubule-kinetochore attachment and chromosome alignment at

the metaphase plate. Treatment with benomyl also results in a decrease in tension at

kinetochores by significantly reducing the distance between the sister kinetochore

pairs. Benomyl also decreases the inter centrosomal distance in mitotic He La cells

and blocks the cells at mitosis. (Rathinasamy et al. 2006). New genes namely, CIN1,

CIN2, and CIN4, affecting microtubule function in Saccharomyces cerevisiae have been identified by screening for mutants that display sensitivity to the antimicrotubule drug benomyl. These genes are also shown to be involved in the common pathway that influence microtubule function. (Stearns et al. 1990). Drugs like carbendazim, thiabendazole, and chloropropham at various concentrations completely arrest cell division along with distinct morphological variations. The cells either became enlarged or they are small and rounded on treatment with carbendazim, and chloropropham respectively. Each of these drugs have different transition points; for example, amiprophos methyl affected a very early stage while carbendazim and thiabendazole block cell division cycle at later stage, indicating that these drugs affect cell cycle in fission yeast either by inhibiting tubulin synthesis (early stage as with amiprophos methyl) or by hindering microtubule assembly (as with carbendazim and thiabendazole). These drugs are useful in studies of microtubule synthesis and their role during the cell division cycle in yeast. (Walker GM, 1982)

CHAPTER III

EXPERIMENTAL PROCEDURE

Using molecular genetics techniques, we have implied following procedures and protocols to continue with this research. The S. pombe strain mut2A has been transformed with the genomic library pTNF2 obtained from Japan Yeast Genetic

Resource using the Lithium acetate protocol. The genomic library was successfully amplified by transforming the compatible E. coli. The ura- trait of mut2a has been utilized for selecting transfromants. Resulting transformants were then replica plated on EMM ALH lacking uracil plates containing 8µgm/ml MBC. Most of the transformants were unable to survive owing to their sensitivity to MBC. Only 0.02% transformant survived indicating that they possibly contain the plasmid with the gene that rescues mut2A strain from MBC damage. The colonies corresponding to these were then picked from original transformants and grid plated on EMM ALH and allowed to grow for 2 days.

To test if the MBC complementation was indeed due to the plasmid and not because of a chromosomal mutation that might have occurred during the transformation procedure, the grid plated colonies were then individually streaked on to YE5S complete media plates to obtain single colonies. The resulting YE5S plates were the again replica plated on to EMM-MBC AULH and EMM-ALH containing media plates. Exact colonies are visible on both the plates. These yeast cells contain the plasmid carrying the gene that is involved in the Rad26 pathway and can be further used to isolate the plasmid which will be then cloned into E. coli and used for sequencing.

Figure 3.1. Gel image of pTNF2 amplification. Restriction digestion of the successful amplification of the pTNF2 genomic library in the E. coli.

Mut2A Transformation

The UV mutagenized S. pombe strain mut2a was transformed by Lithium Acetate protocol using following steps. A single yeast colony growing on YE5S solid media was picked by sterile inoculation loop and inoculated in 50mL of YE5S + 5L carbencillin liquid media in a 200mL flask. The inoculated media was placed in an incushaker at 116 rpm at 30oC for 18 hours. The inoculated culture was transferred to

50ml gene mate tubes and centrifuged on BECKMAN Allegra ZIR centrifuge at

3000rpm for 5 minutes at 4oC. The liquid supernatant was carefully discarded. The pallet was suspended in 1mL of molecular grade water and centrifuged again with the same cycle. The supernatant was carefully decanted, and the pallet was dissolved in the drop of water left and transferred to 1.5mL Eppendorf tube. The cells were then spun for 20 seconds using VNR galaxy mini centrifuge at 3000rpm. The supernatant water was vacuum-sucked to obtain washed pallet. The pallet was mixed with 1mL of

LiAc/TE solution. The cells were recentrifuged for 20 seconds at 3000rpm and supernatant was removed. 10µL of pTNF2 genomic library and 2µL of sheared herring sperm DNA was added to pallet and gently mixed and allowed to incubate at room temperature for 10 minutes. 10µL of these cells were spread on EMM-ALH solid media plates using a flame-sterilized glass spreader to serve as a control. 260µL of 4000 PEG (40% w/v) was added to the reaction tube and placed on incushaker at

30oC, 116rpm for 1 hour. The tubes were intermittently checked to ensure proper mixing. After the completion of 1 hour, 43µL of DMSO was added to the reaction tube, and heat shocked at 42oC for 5 minutes in a heat block. The reaction tube was again centrifuged for 20 seconds at 3000rpm, the supernatant was removed and the pallet was washed with 1mL of EMM-ALH and recentrifuged at 3000rpm for 20 seconds. A final volume of 200µL of EMM-ALH was added to the pallet. 100µL of this final cell volume was spread on EMM-ALH plates using sterile glass spreader and placed in incubator upside down at 30oC for 3 days.

Lithium Acetate solution - This solution was prepared by mixing 8mL of molecular grade water, 1 mL of 10X T.E buffer solution and 1mL 10X lithium acetate solution.

4000 PEG – To prepare a 40% w/v PEG stock solution, 8gm of PEG 4000 was dissolved in 2 mL of 10X T.E, 2 ml 10X LiAc solution and 9.75mL distilled water and the solution was filter sterilized.

Figure 3.2. Transformation of mut2a. Successful transformation of the mut2a with the pTNF2 genomic library on EMM-ALH media. (only one of the successful transformation plate shown)

Replica plating on MBC containing media

The transformants that appeared on the EMM-ALH plates were then replica plated on to EMM-ALH MBC solid media containing 8µgm/mL MBC and 1µgm/mL

PB dye and were incubated at 30oC for three days.

Complementation

The colonies (from original transformant plates) corresponding to surviving ones on MBC media were grid streaked on to EMM ALH media and incubated at 30oC for

2 days. In the next step, each individual colony from grid plate were restreaked on to

YE5S gel media plates to obtain single colonies. The resulting YE5S plates were then again replica plated on to EMM-MBC AULH and EMM-ALH simultaneously.

Figure3.3. Complementation procedure. The steps undertaken to establish the basis of MBC complementation

Genomic DNA extraction

A single colony of the mut2a yeast transformant with the desired plasmid

growing on EMM ALH was picked by a sterile toothpick and inoculated in 1 mL

YE5S media and incubated at 37oC and 150rpm, overnight. The culture was

transferred to 1mL E tube and centrifuged at 14K rpm for 1 minute. The supernatant

was carefully removed, and cells were resuspended in 293µL of 50mM EDTA, 7.5µl

of 75 units/µL lyticase and incubated at room temperature for 1 hour. The reaction

mix was then centrifuged at 14Krpm for 2 minutes, and the supernatant was carefully

pipetted out. 300µl of Nuclei Lysis Solution was added to cell pellet and gently

mixed. Protein Precipitation Solution (100µl of) was added and vortexed vigorously

at high speed for 20 seconds and then incubated on ice for 5 minutes, and then

centrifuged at 14Krpm for 3 minutes. The supernatant was transferred to clean tube

with 300µL of room temperature isopropanol and centrifuged at 14Krpm for 2

minutes. The supernatant was carefully decanted, and 300µLof 70% ethanol was

added to wash the DNA pallet. A final centrifugation was done at 14Krpm for 2

minutes, the ethanol was carefully aspirated, and the pallet was air dried for 10

minutes. DNA was dissolved in 100µL of 1X TE.

Restriction digestion and Gel electrophoresis

2µL of DNA, 16µL molecular grade water, 2µL fast digest buffer were mixed in

the tube that represents the uncut DNA. Similarly, 2µL of DNA, 15.5µL molecular

grade water, 2µL fast digest buffer and 0.5µL BamHI, and 0.5 µL HindIII along with

above-mentioned master mix and both restriction enzyme with master mix were

mixed in separate tubes. All 8 tubes were placed in a thermocycler and run on pre-set restriction cycle.

The following protocol for gel electrophoresis was applied to confirm the presence of genomic DNA purified from the S. pombe. 0.32 gm of Omnipure Agarose was dissolved in 40mL 1X TAE buffer in a 200mL E. flask o prepare a 0.8% solution.

The solution was then microwaved for 45 seconds to ensure complete dissolving of agarose. The solution was then cooled for 1 minute and 4µL of ethidium bromide

(EtBr) was added. After letting this solution cool for about 15 minutes, it was poured in the gel electrophoresis reservoir plated that had been sealed to prevent the running of the solution. The gel electrophoresis comb was placed to punch wells in the gel.

The comb and the seals were carefully removed after the gel was solidified. 1X TAE buffer was then poured into the reservoir to submerge the gel completely.

The restriction reactions were then pipetted in each well of the solidified gel along with 4µL of 1Kb gene ruler ladder. 70mV current was applied to the gel, and it was visualized after 45 minutes in a UV transilluminator and photographed.

Figure 3.4. Gel image of transformed mut2agenomic DNA. Successful purification of DNA (genomic and plasmid) from transformed mut2a that remained viable on MBC media

E. coli Transformation

Transformation of E. coli was done to separate the plasmid of interest from the genomic DNA purified from the yeast. The E. coli competent cells were removed from -80oC freezer and thawed on ice for 20 minutes. 250µL each of the competent cells was put in separate Eppendorf tubes one which serves as negative control. 10µL of the purified genomic DNA was added and incubated on the ice for 10 minutes. The reaction was then heat shocked at 42oC for 2 minutes on the heat block. The cells were then incubated on ice again for 1 minute. 1mL of SOC media was added to reaction mixture which was then transferred to 10mL glass test tube and placed in a shaking bench top incubator set at 37oC and 200rpm for 1 hour. This reaction mix was then transferred to two 1.5 mL E tubes, 100µL from one tube was spread on to LB carb solid media plate with a sterile glass spreader. The remaining reaction was centrifuged at 14K rpm for 1 min. 800µL of supernatant SOC was pipetted out, and cells were resuspended in the remaining SOC. 100µL of cells were then spread on LB carb media plates. The plates along with negative control plate were placed upside down in the incubator set at 37oC for 24 hours.

Alkaline lysis DNA Extraction

Successfully transformed bacterial colonies were picked from the transformation plate and inoculated in separate 1 ml sterile LB plus carbancellin medium using a sterile toothpick and grown overnight in a shaking bench top incubator set at 37oC and 150rpm. The next day cell cultures were transferred to microcentrifuge tube and spun at 14K rpm for 1 minute at room temperature. The supernatant was carefully removed using the pipette. The pallet was resuspended in 100µL GTE solution and incubated at room temperature for 5 minutes. 200µl NaOH/SDS solution was added to

the reaction, and incubated on ice for 5 minutes. A 150µL solution of potassium

acetate is thoroughly mixed in the reaction tube and incubated on ice for 5 minutes.

The reaction was centrifuged at 14K rpm for 1 minute at room temperature. The

supernatant was carefully pipetted into a clean microcentrifuge tube and mixed with

1mL of 95% ethanol, centrifuged at 14Krpm for 2 minutes. The supernatant was

carefully discarded and the microcentrifuge tube containing DNA pallet was air dried

to evaporate all the alcohol. The plasmid DNA was dissolved in 100µL of 1X TE.

Gel electrophoresis

Gel electrophoresis was performed to confirm the presence of plasmid DNA and

determine the size of the insert DNA in the plasmid. 2µl plasmid was mixed with

restriction digestion solution containing 2µl fast digest buffer premixed with dye,

0.5µl restriction enzyme and 15µl of molecular grade water.

0.8% gel was prepared by dissolving 0.32 gm of Omnipure Agarose in 40mL 1X

TAE buffer in a 200mL E. flask. The reagents were microwaved for 45 seconds to

ensure thorough dissolving of agarose. The solution was then allowed to cool for 1

minute then 5µL of ethidium bromide (EtBr) was added. The solution was allowed to

cool for about 15 minutes, and poured in the sealed gel electrophoresis reservoir plate

to prevent the running of the solution. The gel electrophoresis comb was used to

punch wells in the gel. The comb and the seals were carefully removed after the gel

was solidified. 1X TAE buffer was then poured into the reservoir to submerge the gel

completely.

The restriction reactions were then pipetted in the solidified gel wells along with

4µL of 1Kb gene ruler ladder. 70mV current was applied to gel, and it was visualized after 45 minutes in a UV transilluminator and photographed.

MIDI Prep for Vector and insert size determination

A midi prep reaction was performed by Qiagen midi-prep kit to get a better yield

of the plasmid DNA so it can be used to estimate the size of the DNA insert in the

plasmid.

Restriction digest

The restriction digest reactions were performed on the plasmid DNA by making

a master mix containing 60µL molecular grade water, 8µL of fast digest buffer. 17µL

each of this reaction mix was added to four separate thermocycler tubes which already

contained 2 µL each of plasmid DNA. 0.5µL ClaI was added to one tube, 0.5µL SphI

to other, and 0.5µL each of ClaI and SphI was added. The last tube was used without

restriction enzyme to serve as an uncut plasmid reference. The reaction tubes were

then placed in the thermocycler at preset restriction digest cycle for 5 hours.

Gel Electrophoresis

Gel Electrophoresis was performed using the restriction digest reaction

according to the previously described protocol to deduce the size of the insert.

Primer design

Both the forward and reverse sequencing primers were designed based on the vector

pBR322 backbone of the pTNF2 genomic library. The 927 S. pombe strain genomic

DNA was cleaved with BamHI and ligated to the same site in the vector. Therefore, pBR322 sequence upstream of the BamHI site was used for the forward sequencing primer and pBR322 sequence downstream was used for reverse sequencing primer.

Based on these references following primers were designed and ordered from

Integrated DNA Technologies.

Forward primer- 5’ GGC GAC CAC ACC CGT CCT GTG 3’

Reverse primer- 5’ GCG TCC GGC GTA GAG GAT C 3’

Sequencing reaction mix

The concentrations of the primers and the midi prep plasmid DNA were measured using the Nano drop 2000 which returned the following results-

Forward primer concentration 447.9ng/µL

Reverse primer concentration 924.1ng/µL

Plasmid concentration 356.6 ng/µL

All the nucleotides were then diluted to final concentrations required by the

Biofunctional, service used for sequencing.

Transformation with genes present in sequenced plasmid

The mut2a was individually transformed following the above-described protocol with the pap1 clone, the hrp1 clone ordered from RIKEN Japan along with pTNF2 and sequenced library plasmid that served as controls. Before transformation, each of the DNA was restriction digested with NdeI and HindIII to check for degradation.

Figure 3.5. Gel image confirming DNA integrity.

Replica plating

Each of the above-described transformant was then replica plated on to EMM

ALH media plates with 8µgm/mL concentration of MBC.

Sequencing of pap1 gene from mut2a

We designed the following random primers to sequence the entire pap1 gene from the mut2a to identify the mutation.

Figure 3.6. Primers for pap1 sequencing. Detailed description of the primers (aligned to their sequence in the gene) used for the sequencing of the pap1 gene from mut2a

CHAPTER IV

RESULTS

Out of approximately 20,000 colonies of mut2a successfully transformed with the pTNF2 genomic library, only a 0.02% of transformants survived on to MBC solid media indicating the presence of the plasmid containing the gene conferring MBC resistance.

Figure 4.1. Transformed mut2a replica plating. Mut2A transformants replica plated on to EMM ALH MBC were non-viable due to an absence of plasmid conferring MBC resistance.

Figure 4.2. Replica plate highlighting viable colony. Transformation and replica plating results of mut2a on to selective media showing single viable colony on the EMM ALH MBC media and the corresponding colony on EMM ALH media.

Complementation

To establish that the MBC complementation was in fact due to the presence of

episomal plasmid and not because of any other possible chromosomal mutation, the

colonies (from original transformant plates) corresponding to surviving ones on MBC

media were grid streaked on to EMM ALH media and incubated at 30oC for 2 days. 28

We used this step to establish selection pressure and made sure that the cells

maintained the plasmid. Streaking of the colonies on to YE5S non- selective media

allowed the growth of all the cells irrespective of the genotype. Again, the replica

plating on to EMM AULH MBC and EMM ALH media and comparing the plates

ensure that only the colonies with the desired plasmid with ura gene marker will be

viable and be growing on EMM ALH. If the initial phenotype was due to mutation,

the growth on EMM ALH media would be absent due to lack of plasmid.

Figure 4.3. Plasmid complementation. All the same colonies are growing on EMM AULH MBC, and EMM ALH confirming that complementation is indeed due to an episomal plasmid. (highlighted colonies are ones that were growing on YE5S and EMM AULH MBC media but not on EMM ALH confirming absence of plasmid)

Determination of the size of insert in the plasmid

The restriction digestion of the isolated plasmid with BamHI resulted in single

fragment confirming the presence of one Bam HI site while digestion with Hind III

gave rise to 2 fragments of 1000bp, 1 of approximately 2500bp, and larger fragment

estimated to be about 11000bp. Use of the restriction enzymes in combination yielded

5 fragments measuring 2 of 1000bp, 1 of 2500bp and one each of 6000bp and approx.

4500bp. Based on these measurements we estimated the overall size of the plasmid along with insert DNA to be approximately 15000bp to 15500bp.

Figure 4.4. Restriction digestion of the plasmid. Restriction digestion and gel electrophoresis of the plasmid to determine the size of plasmid

Referring to the estimated size of the plasmid we utilized Cla I and Sph I to estimate the size of the DNA insert in the plasmid. Digestion with ClaI produced a single fragment of 9500bp, two of approximately 3000bp and one around 1800bp.

SphI digestion produced 11000bp and 6000bp. Combination of both the restriction enzyme resulted in a 6000bp fragment, two 3000bp fragments, two fragments of

2000bp each and single fragment of 1000bp. Based on this information and comparing the position of these restriction sites in pBR322 vector backbone, the size of the insert DNA is calculated to be approximately 8160 bp whereas the entire size of the plasmid comes around approximately 17000bp.

Figure 4.5. restriction digestion of the plasmid conferring MBC resistance. Restriction digestion and gel electrophoresis of the sequenced library plasmid.

Figure 4.6. Vector with estimated insert size.

Sequencing results and Nucleotide alignment

The results obtained from the Biofunctional services were aligned through

BLAST from NCBI which pointed towards the presence of four candidate genes hrp1,

atg12, pap1, and rpl1502 and an uncharacterized Zn finger domain on the plasmid

conferring MBC resistance found on the chromosome 1 of S. pombe.

https://www.pombase.org/gene/SPAC1783.07c Figure 4.7.Alignment results. Nucleotide alignment result of the sequenced plasmid showing hrp1, atg12, pap1, and rpl1502

We believed either hrp1 or pap1 was the candidate gene imparting desired characteristics. Each of these gene clones was ordered from Japan Yeast genetic resources and further utilized for the transformation of mut2a and replica plating of the transformants on to MBC media along with the pTNF2 and the sequenced library plasmid to serve as controls. The results substantiated the role of pap1 in conferring

MBC resistance owing to the growth and light pink color of cells.

Hrp1 Pap1 Sequenced library plasmid

Figure 4.8 Replica plating results with pap1, hrp1, and sequenced library plasmid. Comparative image of the replica plating results of the mut2a transformed with pap1, hrp1 and sequenced library plasmid.

Sequencing of pap1 from mut2a

To further substantiate our finding, we sequenced the pap1 from the rad26:4A

UV mutagenized cells. The results showed the absence of the mutation in the original pap1 gene. This lead us to believe that expression of extracopies pap1 confers MBC resistance.

CHAPTER V

DISCUSSION

To identify the protein that interacts with Rad26 during the interphase checkpoint response to microtubule damage, we transformed mut2A with the ura+ marked pTNF2 genomic library and selected transformants on media without uracil.

Ura+ transformants were then replica plated onto MBC media. Of 20,000 transformants, the viable colony was identified. Replica plating of this colony showed that MBC-resistance was conferred by an autonomously replicating ura+ marked plasmid from the library and therefore was not the result of a second site genomic suppressor. The plasmid was isolated, and the sequencing results of the plasmid determined the presence of hrp1, pap1, atg12, rpl1502 and an uncharacterized

ORF with a zinc finger domain. Repetition of the above procedure with clones containing only one of each of the genes listed above showed that only pap1 confers

MBC resistance in the mut2a strain. If pap1+ was allelic to mut2a, then sequencing of pap1 should have revealed a mutation. However, the sequencing results showed that the pap1 gene from the mut2A strain was not defective. These results indicate that

MBC resistance of mut2a is possibly due to the expression of extra copies of pap1 from the plasmid in addition to inherent cellular pap1 as opposed to the interaction between rad26 and pap1. This phenomenon is called extra-copy suppression and usually occurs following overexpression of downstream components of signal transduction pathways that bypass requirements for upstream components.

The pap1 is an AP-1 like transcription factor with a bZip domain that regulates the transcription of various genes that are required for cellular stress responses. The bZip domain is required for binding to DNA with high affinity and specificity. Pap1 binds to conserved cis-regulatory motifs that are in proximity to the target genes to 34

regulate their transcription. For example, pap1 upregulates the transcription of catalase enzymes in response to oxidative stress. It is also involved in activation of promotors necessary for cellular multidrug resistance.

Pap1 and its homolog of budding yeast Yap1 is localized in the cytoplasm during normal conditions, but they are translocated to the nucleus in response to stress. The regulation of stress-induced translocation is dependent on redox reaction of two cysteine-rich domains (CRD) present in Yap1 as well as Pap1. (Toone et al.

1998). They have also shown that stress-induced nuclear regulation of pap1 is also dependent on MAP Kinase sty1.

Substantial evidence has been provided by various studies on the activation of stress-induced Mitogen-activated protein kinase (MAPK) pathways in mammals, budding yeast, and Drosophila. Pap1 and human c-Jun protein share similarity with respect to their structure and DNA binding properties. The homology of pap1 to the mammalian c-Jun emphasizes conservation of similar stress-induced mechanism in fission yeast and human. MAPKs are serine-threonine proteins, and essential components of stress-induced signal transduction pathway that sense and communicate extracellular stress signal through the cytoplasm to the nucleus. Several bZip containing DNA binding transcription factors like pap1 are the downstream targets for these MAP kinases. For example, Sty1 is a crucial component of fission yeast MAPK pathway and share 50% similarity to human p38 MAP kinase. Pap1 (in addition to Atf1) is the downstream target of Sty1 in response to multidrug and oxidative stress that directly regulate transcription of relevant genes essential for stress response.

Several studies have also highlighted the role of pap1 human homolog c-Jun in cell cycle progression. Overexpression of c-Jun results in a more significant

proportion of cells in S, G2, and M as compared to G1 (interphase). This response of

c-Jun occurs through the transcriptional activation of cyclin D1 (cdk regulator) and

deactivation of cdk inhibitor p21. Based on pap1 and c-jun homology, it might be

suggested that over expression of pap1 in mut2a possibly manipulates the cdk

expression or other downstream genes required for cell viability and progression of

cell cycle in response to MBC insult.

Study with microtubule inhibiting drugs in human cell lines suggested that

microtubule destabilization results in distinct MAP kinase signaling and cross-talk

between them determine the final cellular response. Treatment with microtubule

destabilizing drugs resulted in significant increase in the MAP kinase family member

JNK (with inactivation of its antagonist ERK) whose downstream target Jun-c and

other kinases coordinate and mediated signaling pathway required for appropriate

cellular response for microtubule maintenance. Some studies show that JNK signaling

leads to apoptosis while others claim to have protective action depending on the

activation of downstream targets. For example, MEKK1, a component of MAPK

pathway selectively regulates c-Jun kinase to prevent apoptosis and increase cell

survival in case of microtubule damage. The c-Jun homology with pap1 possibly

signifies the existence of similar mechanisms in fission yeast. (Leppä et al. 1999)

Multiple studies of the stress response mechanism have identified pap1 and its

downstream target genes associated with response mechanism. However, the exact

role of pap1 in MBC induced microtubule damage in mut2A warrants further

elucidation. A mechanism in response to G2 phase microtubule poisoning with MBC

has been demonstrated by Balestra et al. (2008) by analyzing the relationship between the integrity of microtubules and cell cycle control. Evidently, during interphase, MTs organize into spindle assembly which is required for chromosome separation. The

treatment with MBC depolymerizes the microtubules hindering assembly into spindle that disturbs chromosome separation. They established that in the above-described case the checkpoint kinase Wee1 influence the cell cycle by phosphorylation and inhibition of cdc2. The inhibition of cdc2 eventually results in cell cycle arrest and provide an opportunity for the damage repair. It may be suggested that pap1 directly downregulates the expression of cdc2 leading to cell cycle arrest. An alternate hypothesis can be suggested on possible activation of MAPK in response to MBC stress resulting in pap1 dependent transcriptional upregulation of checkpoint effector kinases like wee1 that inhibit cdc2 or similar downstream targets of cell cycle checkpoint that arrest cell cycle until the damage to microtubule is repaired.

Toda et al. (1991) showed that over-production of pap1 expressed from multicopy plasmid confer resistance to staurosporine, a protein kinase inhibitor. They hypothesized that either pap1 upregulates or activates the kinases that are targeted by the drug or inactivation of the kinases by drug treatment initiate a feedback mechanism that leads to activation of the same kinases in pap1 dependent manner.

These hypotheses may be applied to MBC rescue where pap1 possibly upregulates tubulin expression that promotes microtubule polymerization.

Genetic screens for caffeine resistance (Calvo et al. 2009) in fission yeast have shown that pap1 upregulates the transcription of genes coding for the efflux pumps.

Up-regulation of efflux pump has also been associated with resistance to multidrug and is a widely-accepted pap1 induced resistance mechanism.

CHAPTER VI

CONCLUSION

Based on the findings of the above-described experiments we report that expression of extra copies of the pap1 rescue mut2a from MBC stress. This is a novel role of pap1 extra-copy expression in compensating MBC stress.

Stress-induced nuclear localization of pap1 and a downstream target of MAPK stress response strongly suggest the possibility of the over expressed Pap1 localizing to the nucleus in response to MBC and triggering S. pombe rescue through a MAPK related yet unidentified mechanism.

Extra copy Pap1 induced resistance against genotoxic drugs further corroborate our finding. Belfield et al. (2014) identified extra copies of pap1 expressed from multi-copy plasmid conferring DNA damaging drug resistance in chk1 deletion and ckh1 mutant cells. Pap1 confers viability in chk1 deletion cells suggest that chk1 and pap1 function independent of each other indicating that this pap1 rescue does not delay cell cycle progression. This observation contradicts our suggestion that pap1 rescue involves transcriptional regulation of cell cycle- associated genes. As the pap1 induced MBC rescue mechanism is not yet identified this idea cannot be disregarded entirely. Although, mechanism aiding over-expressed pap1 in drug resistance is hypothesized to be the upregulation of cellular efflux pumps that flush out the drug from the cell, the process involved in DNA and microtubule damage resistance is yet to be determined.

The identity of the mut2a gene remains unresolved. Further attempts to clone it using the genomic complementation procedure outlined in this thesis need to be performed.

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APPENDIX

Acronyms

ATM- Ataxia Telangiectasia Mutated

ATR- Ataxia Telangiectasia Mutated and Rad3 Related

ATRIP- ATR Interacting Protein

BamHI- Bacillus amyloliquefaciens H 1

BRCA1-Breast-ovarian cancer susceptibility 1

Chk1- Checkpoint Kinase 1

DDR- DNA Damage Response

G1- Gap 1

G2- Gap 2

G1-S- gap1-Synthesis

G2-M- Gap2-Mitosis

GTP- Guanosine triphosphate

HeLa- Henrietta Lacks

MAPK-Mitogen-activated protein kinase

MBC- Methyl 2-benzimidazolecarbam

MT- Microtubule

MTOCs- microtubule-organizing centers

Nde1 - NADH Dehydrogenase, External I

NES -Nuclear Export Signal

PCM- Pericentriolar matrix of proteins

PI3Ks- Phosphatidyl Inositol 3-kinases

RPA -Replication Protein A

SPB- Spindle Pole Body

PCNA- Proliferating cell nuclear antigen

TBZ- Thiabendazole

SS-Seckel syndrome

UV-Ultaviolet