A study on premature segregation of unreplicated chromosomes 1

104 4.2.1 Premature segregation of unreplicated chromosomes in cells lacking Cdc7 and Cdc45……… 104 4.2.2 Depletion of Cdc6 in cdc34-1 cells fails to promote spindle assembly or spindle

2011

A STUDY ON PREMATURE SEGREGATION OF

UNREPLICATED CHROMOSOMES

KHONG JENN HUI

B Sc (Hons.), MURDOCH UNIVERSITY

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF

PHILOSOPHY

INSTITUTE OF MOLECULAR AND CELL

BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2011

Acknowledgements

I would like to express my earnest thanks, sincere gratitude and appreciation to Professor Uttam Surana for his guidance, insightful and stimulating discussions, as well as valuable advice, which helped sustain my curiosity and passion in this study

My sincere thanks also go out to members of my PhD Supervisory Committee, A/P Yang Xiaohang (IMCB) and Dr Maki Murata-Hori (TLL), for their constructive comments and encouragement

My deepest gratitu de to Assistant Prof Lim Hong Hwa, Dr Zhang Tao and Dr Sihoe SanLing for your continuous help, discussions, guidance and advice, without which this project and thesis would never have become a reality

Special thanks to my lab mates – Dr Yio WeeKheng, Dr Idina Shi Yiting, David, HongQing, Joan and all members of CMJ and WY laboratory for sharing, discussions and generous help in various ways

I would like to thank Dr Jayantha Gunraratne and Associate Professor Walter Blackstock (IMCB) for the collaboration on mass spectrometry

I am grateful to Drs Kim Nasmyth, Frank Uhlmann, Tomo Tanaka, Piatti Simonetta, Matthias Peter for providing me with valuable reagents, yeast strains and constructs which were essential for many experiments

Most importantly, I wish to thank my parents, brother and sister, for their unconditional support, encouragement, prayers and advice Last but not least, I would like to extend my gratitude to my wife kilyn for your understanding, patience, support and encouragement throughout this study

Table of Contents

Acknowledgements……… i

Table of Contents……… ii

Summary………vi List of Tables……… ix

List of Figures……… x

List f Symbols……… … xiii

Chapter 1 Introduction….………….……… 1

1.1 Introductory Remarks……… 1

1.2 Brief overview of cell cycle……… 2

1.2.1 Saccharomyces cerevisiae cell cycle……… 2

1.2.2 Regulation of the transition point between cell cycle phases and cyclin-dependent kinase Cdc28 5

1.2.3 Regulation of Cdk activity 6

1.2.4 Regulation of cell cycle events by checkpoints 7

1.3 Regulation of cell cycle by protein degradation 8

1.3.1 The ubiquitin-proteasome system…….………9

1.3.2 SCF……… 10

1.3.2.1 SCFCdc4……… ……… 11

1.3.2.2 SCFGrr1……… 13

1.3.2.3 SCFMet30 14

1.3.3 APC……… 14

1.3.3.1 Substrate specificity of APC……… 15

1.3.3.2 Regulation of anaphase by APC……….17

1.3.3.3 Regulation of mitotic exit by APC……… 18

1.3.3.4 Regulation of G1-S by APC……….……… 19

1.4 The spindle pole body cycle………… ……… 21

1.4.1 Spindle Anatomy……… 24

1.4.2 Regulation of microtubule dynamics……… 27

1.4.3 Regulation of short spindle formation……… 32

1.4.4 Regulation of spindle elongation……… 34

1.5 Premature spindle elongation and segregation of unreplicated

chromosomes………36

1.6 The G1-M checkpoint pathway………39

1.7 The multiple roles of Cdc6……… 40

Chapter 2 Materials and Methods……….……… 50

2.1 Materials……… 50

2.2 Methods……… 50

2.2.1 Escherichia coli strains and culture conditions……… 62

2.2.2 Yeast strains and culture conditions……… 62

2.2.3 Cell cycle synchronization……… 63

2.2.4 Yeast transformation………64

2.2.5 Isolation of plasmid DNA from yeast……… 64

2.2.6 Yeast chromosomal DNA extraction……… 65

2.2.7 Southern blot analysis……… …… 66

2.2.8 Immunofluorescence staining (IF)……….……… 67

2.2.9 Microscopy……… 68

2.2.10 Flow cytometry analysis (FACS)……….69

2.2.11 Preparation of cell extracts for protein analysis………70

2.2.11.1 Protein extraction using Tri-Chloroacetic Acid (TCA)…… 70

2.2.11.2 Protein extraction using acid-washed glass beads………… 70

2.2.12 Western blot analysis……… 71

2.2.13 Immunoprecipitation………71

2.2.14 PCR-based strategy for fluorescent protein and epitope tagging of yeast genes……… 72

2.2.15 Pulse-chase assay……….73

2.2.16 Sample preparation for SILAC mass spectrometry………….73

Chapter 3 Premature chromosome segregation in cells with unreplicated chromosomes ………75

3.1 Background……… 75

3.2 Results……… 79

3.2.1 Cells depleted of Cdc6 undergo premature nuclear division in the absence of DNA replication……… 79

3.2.2 Premature nuclear division in Cdc6 depleted cells is associated with major mitotic events……….82

3.2.3 Precocious nuclear division in Cdc6 depleted cells does not require onset of mitosis………86

3.2.4 Precocious nuclear division in Cdc6 depleted cells can be prevented by dicentric chromosomes……… 91

3.2.5 Precocious nuclear division in Cdc6 depleted cells is due to deregulation of spindle dynamics………94

3.3 Discussion……….……… 99

Chapter 4 Regulation of spindle elongation by Cdc34……… 103

4.1 Background……… 103 4.2 Results……… 104 4.2.1 Premature segregation of unreplicated chromosomes in cells

lacking Cdc7 and Cdc45……… 104 4.2.2 Depletion of Cdc6 in cdc34-1 cells fails to promote spindle

assembly or spindle elongation……….… 109 4.2.3 Ectopic expression of Sic1 and Cdh1 prevent premature spindle

elongation in Cdc6 depleted cells……… 111 4.2.4 cdc34 and cdc34 cdc6 mutant cells can assemble short bipolar

spindles in the absence of Cdh1 or Sic1 but fail to elongate them……… 114 4.2.5 Sic1 degradation promotes Cdh1 inactivation and short spindle

assembly……… 117 4.2.6 Ectopic expression of microtubules associated proteins induces

spindle elongation in Cdc34 deficient cells devoid of Cdh1……… 120 4.2.7 Cdh1 resistant microtubule associated proteins cannot induce

complete spindle elongation in cdc34-1 and cdc34-1 cdh1Δ

cells……… 124 4.2.8 Loss of Ase1 or Cin8 individually cannot prevent premature

spindle elongation in Cdc6 deficient cells……… 127 4.2.9 Cdc34 can induce spindle elongation by promoting stability of

microtubule associated proteins……… 129 4.2.10 Microtubule associated proteins Ase1 and Cin8 are unstable in

cells deficient in Cdc34……… 137 4.2.11 Cdc34-mediated stabilization of microtubule associated proteins

are proteasome dependent……… 140 4.3 Discussion……… … 142

Chapter 5 A search for Cdc34-mediated up- or down regulated

proteins that promote spindle elongation……… … 147 5.1 Background……… 147 5.2 Results……… 148 5.2.1 Cdc34 promotes up-regulation of the polo-like kinase Cdc5

during premature spindle elongation……… 148 5.2.2 Ectopic expression of Cdc5 can induce spindle formation and

elongation……… 152 5.2.3 Cdc5 is unstable in the absence of Cdc34 function…… 154 5.3 Discussion……… 156

Chapter 6 Perspective and future directions……… 158

Summary

High fidelity transmission of the genome to the next generation is imperative for the successful survival of all species At cellular level, this can be accomplished by accurate duplication and segregation of the genome to two daughter cells during the cell division cycle In order for chromosome segregation to proceed accurately, the sister chromatids must be attached to the mitotic spindle Hence, cells have evolved surveillance pathways known as checkpoints to ensure that both spindle cycle and cell cycle progress in a coordinated and timely manner These checkpoints halt cell cycle progression when damage or defect is detected on chromosomes or spindles and undertake immediate steps to repair detected damages before the cell cycle is allowed

to resume This cell cycle halt can pose extreme danger to cell cycle committed cells (post START) that cannot initiate S phase because spindle forms in the absence of duplicated chromosomes and biorientation will lead to precocious chromosome segregation and genomic instability, a leading cause of aneuploidy

Both DNA Replication and damage checkpoints are known to prevent precocious spindle elongation (i.e premature chromosome segregation) via regulation

of spindle dynamics (Krishnan et al 2004) (Zhang et al 2009) A less characterized checkpoint known as the G1-M checkpoint has also been reported to play essential role in prevention of mitosis should cells fail to undergo S phase Cdc6 has been defined as an important component of the G1-M checkpoint that prevents untimely onset of mitosis when cells fail to initiate DNA replication This is because yeast cells deficient in Cdc6 fail to initiate DNA replication but proceed to elongate their spindles and segregate the un-replicated chromosomes leading to a “reductional” anaphase (Piatti et al 1995) Due to the intimate association between chromosome

segregation and mitosis, it has been proposed that Cdc6 or the G1-M checkpoint prevents onset of mitosis when cells fail to initiate DNA replication (Piatti et al 1995) Thus far, no other component of this checkpoint pathway has been identified

In Chapter 3, our results suggest that untimely chromosome segregation in the absence of Cdc6 function is not due to premature mitotic entry but is a result of the deregulation of spindle dynamics Surprisingly, we also find that premature chromosome segregation is a not a property specifically associated with the loss of Cdc6 function but it is a common characteristic of cells (such as cdc7 or cdc45 mutants) that fail to initiate DNA replication

In Chapter 4, our findings implicate Cdc34 (SCF) as a new regulator of spindle dynamics The clue came to light from the experiment in which spindles were

dramatically extended in cdc34-1 cdc6Δ cells when Cdc34 function was restored by a

return to the permissive temperature (Figure 27 and 28) This clearly suggests that Cdc34 function is necessary to convert a short spindle to a long spindle and argues

that cdc6 mutant cells require Cdc34 function to extend their spindles

In conclusion, the dramatic deregulation of spindle dynamics experienced by cells that are committed to the cell cycle but fail to undergo DNA replication is a result of the interplay of four sequential cellular events: activation of the E3 ubiquitin ligase SCF, destruction of Cdk inhibitor Sic1, inactivation of another ubiquitin ligase APCCdh1and stabilization of microtubule associated proteins The role of Cdc34 in spindle dynamics is particularly critical during the period between START and S phase in that Cdc34-mediated stabilization of Ase1, Cin8 and Cdc5 (or destabilization

of a novel spindle-elongation inhibitor) would cause premature spindle elongation in any cell that traverses START but are unable to initiate S phase These results suggest

that initiation of DNA replication saves the cells from potential segregation catastrophe

chromosome-List of Tables

Table 1 Reagents used in this study……… 50 Table 2 Antibodies used for immunofluorescence and protein analyses… 50 Table 3 List of S cerevisiae strains used in this study……… 51

Table 4 List of plasmids used in this study……… 57

Table 5 List of the main oligonucleotides used in this study……… 59 Table 6 Cdc34 mediated up- and d-regulated proteins with functions relevant to

SCF and spindle dynamics……… 149

List of Figures

Figure 1 Schematic diagram of the budding yeast cell division cycle… 4

Figure 2 The Spindle Pole Body (SPB) duplication cycles……… 23

Figure 3 The spindle anatomy……… 26

Figure 4 Regulation of microtubule dynamics……… 31

Figure 5 Schematic diagram of Cdc6 protein……… 44

Figure 6 A model for the role of Cdc6 in regulating the activity of

APCCdc20 in anaphase……… 47

Figure 7 Cells depleted of Cdc6 undergo premature nuclear division in the absence of DNA replication……… 81

Figure 8 (A) Premature nuclear division in Cdc6 depleted cells is associated with major mitotic events……… 84

(B) Premature nuclear division in Cdc6 depleted cells is associated with major mitotic events……… 85

(C) Premature nuclear division in Cdc6 depleted cells is associated with major mitotic events……… 85

Figure 9 APC activity is not required for the precocious chromosome segregation in Cdc6 depleted cells……… 88

Figure 10 APC activity is not required for the precocious chromosome segregation in Cdc6 depleted cells……… 90

Figure 11 Precocious nuclear division in Cdc6 depleted cells can be prevented by dicentric chromosomes……… 93

Figure 12 Precocious nuclear division in Cdc6 depleted cells is due to deregulation of spindle dynamics……… 96

Figure 13 Precocious nuclear division in Cdc6 depleted cells is due to deregulation of spindle dynamics……… 97

Figure 14 Precocious nuclear division in Cdc6 depleted cells is due to deregulation of spindle dynamics……… 98

Figure 15 Premature segregation of unreplicated chromosomes in cells lacking Cdc7……… 107

Figure 16 Premature segregation of unreplicated chromosomes in cells lacking Cdc45……… 108

Figure 17 Depletion of Cdc6 in cdc34-1 cells fails to promote spindle assembly or spindle elongation……… 110

Figure 18 Ectopic expression of Sic1 and Cdh1 prevent premature

spindle elongation in Cdc6 depleted cells……… 113 Figure 19 cdc34 and cdc34 cdc6 mutant cells can assemble short

bipolar spindles in the absence of Cdh1 or Sic1 but fail to

elongate them……… 116 Figure 20 Regulatory scheme outlining the connection between

Sic1 and Cdh1 for the regulation of spindle dynamics

in cdc6 mutant……… 117

Figure 21 Sic1 degradation promotes Cdh1 inactivation and short

spindle assembly……… 119 Figure 22 Ectopic expression of microtubule associated proteins

induce spindle elongation in Cdc34 deficient cells devoid

of Cdh1……… 122 Figure 23 Ectopic expression of microtubule associated proteins

induce spindle elongation in Cdc34 deficient cells devoid

of Cdh1……… 123 Figure 24 Cdh1 resistant microtubule associated proteins cannot

induce complete spindle elongation in cdc34-1 and

cdc34-1 cdh1∆ cells……… 125

Figure 25 Cdh1 resistant microtubule associated proteins cannot

induce complete spindle elongation in cdc34-1 and

cdc34-1 cdh1∆ cells……… 126

Figure 26 Loss of Ase1 or Cin8 individually cannot prevent

premature spindle elongation in Cdc6 deficient cells… ……… 128 Figure 27 Cdc34 can induce spindle elongation by promoting

stability of microtubule associated proteins……… 132 Figure 28 Cdc34 can induce spindle elongation by promoting

stability of microtubule associated proteins……… 134 Figure 29 Cdc34 can induce spindle elongation by promoting

stability of microtubule associated proteins……… 136 Figure 30 Microtubule associated proteins Ase1 is unstable in cells

deficient in Cdc34……… 138 Figure 31 Microtubule associated proteins Ase1 is unstable in cells

deficient in Cdc34……… 139

Figure 32 Cdc34-mediated stabilization of microtubule associated

proteins are proteasome dependent……… 141 Figure 33 Cdc34 promotes up-regulation of the polo-like kinase Cdc5

during premature spindle elongation……… ……… 151 Figure 34 Ectopic expression of Cdc5 can induce spindle formation

and elongation……… …… 153 Figure 35 Cdc5 is unstable in the absence of Cdc34 function……… 155 Figure 36 The emerging model proposed that Cdc34 has a dual

role in mediating premature spindle elongation in cells

that fail to undergo S phase……….… 163

DNA Deoxyribonucleic acid

DIC Differential interference contrast

DNA Deoxyribonucleic acid

PCR Polymerase Chain Reaction

PEG Polyethylene glycol

PMSF Phenymethylsulfonylfluoride

Raff Raffinose

RNA Ribonucleic acid

SDS Sodium dodecyl sulphate

TE Tris-EDTA buffer

TRP Tryptophan

ts Temperature sensitive

URA Uracil

YEP Yeast extract peptone

YEPD Yeast extract peptone + glucose

Chapter 1 Introduction

1.1 Introductory Remarks

Living organisms, uni- or multicellular, perpetuate their respective species through their capacity to reproduce While sexually reproducing multicellular organisms course through a series of developmental stages and require a willing partner before they can produce a progeny, unicellular organisms (or cells that make up multicellular organisms) multiplies via cellular division To give rise to two cells from one, cells undergo a series of ordered cellular events (collectively known as cell division cycle

or simply cell cycle) during which chromosomes are faithfully duplicated and accurately partitioned into the two prospective daughters The orderly progress through the cell cycle is coordinated by two sets of controls: (i) One that enforces an interdependence between major events such as spindle formation, chromosome replication, spindle elongation, chromosome segregation and cytokinesis, and (ii) those enforced by surveillance systems known as checkpoint controls to ensure that the initiation of a later event is prohibited if a prior event is erroneous or incomplete These checkpoints arrest cells at specific stages of the cell cycle when defects are detected, thus allowing sufficient time to repair the errors before the cell cycle can be resumed This process must be tightly regulated and coordinated to prevent accumulation and transmission of harmful lesion to the progeny cells - lesions that can lead to genomic instability and aneuploidy often found associated with cancers (Thompson et al 2010)

Much of the current knowledge relating to the core control networks that govern eukaryotic cell cycle come from the experimental findings using simpler

organisms such as budding yeast Saccharomyces cerevisiae and fission yeast

Schizosaccharomyces pombe, due to their amenability to genetic manipulation

Despite the fact that yeasts undergo close mitosis (in which the nuclear membrane remains intact during mitosis) and vertebrate cells pursue open mitosis (where the nuclear membrane breaks down), the control circuits that regulate cell division are highly conserved among them despite a large evolutionary distance (Byers 1981) Therefore, in this study, we have used budding yeast as an experimental system to investigate the mechanisms that underlie a curious but detrimental cellular behaviour: yeast cells that fail to undergo DNA replication (S phase) become insensitive to controls that coordinate cell cycle events, proceed to prematurely segregate the unreplicated chromosomes and rapidly lose viability An exploration of such extreme behaviour can be instrumental in understanding the nature of the control circuits that normally regulate cell division cycle but lead to ‘pathological manifestations’ when cells are exposed to higher stress-loads Before embarking on a discussion of checkpoint controls and events leading to premature chromosome segregation, it is useful to begin with a brief description of the general regulatory landscape of the cell division cycle to set the stage

1.2 Brief overview of cell cycle

1.2.1 Saccharomyces cerevisiae cell cycle

The budding yeast Saccharomyces cerevisiae cell cycle has been studied extensively

and is best characterized among all eukaryotes Similar to other eukaryotes, cell division in budding yeast is accomplished by the coordinated control of four phases (G1, S, G2, M) (Figure 1) In G1 phase, a cell prepares itself for a new division cycle

by accumulating sufficient resources while continuing to grow to a specific size This

is followed by S phase where the genome is duplicated The cell then continues to grow in size while preparing itself for entry into mitosis during the short G2 phase (approximately 3 minutes in a 90-120 minutes’ division cycle) A successful cell division can only be attained when the duplicated sister chromatids are segregated equally between the mother and daughter cells in M phase (mitosis) Broadly, the M phase is divided into four sub-phases; prophase, metaphase, anaphase and telophase

It is essential that duplicated chromosomes achieve biorientation and congress to the metaphase plate During anaphase, dissolution of sister chromatid cohesion (anaphase A) occurs followed by dramatic elongation of mitotic spindle (anaphase B) which is associated with chromosome segregation towards the two opposite spindle poles – this last event being mediated by the mitotic spindle in telophase stage Finally, cells exit from mitosis followed by cytokinesis, during which the mother and daughter cells separate physically into two independent entities

Figure1: Schematic diagram of the budding yeast cell division cycle

The cell division cycle of Saccharomyces cerevisiae is divided into four phases:

G1, S, G2 and M Passage through START (indicating irreversible commitment

to a new cell cycle division) at late G1 requires the activation of the G1-kinase complexes and marks the commitment of the cells to a new division cycle The emergence of a bud and duplicated SPBs mark the entry into S phase The S- phase kinase complexes trigger DNA replication At late S phase, SPB separation occurs, leading to assembly of short bipolar spindle Activation of the mitotic kinase complexes is pivotal for spindle elongation, chromosome segregation and other key events in mitosis The major destruction machinery that acts during the cell cycle (SCF and APC) is also depicted in the diagram

1.2.2 Regulation of the transition point between cell cycle phases and cyclin-dependent kinase Cdc28

The four discrete phases of cell cycle are tightly regulated and coordinated but are not merely temporally organized series of intervals The different transition points such

as START (indicating irreversible commitment to a new cell cycle division),

G1-S, S-M (entry into mitosis) and M-G1 (mitotic exit) are dependent on the integration

of signal transduction systems that are activated by external signaling molecules (such

as growth factors) and various intracellular cues (eg., damage of cell constituents) (Hulleman et al 2001); (Boonstra 2003); (Hartwell et al 1977) In response to the integrated cues of these signaling pathways, the cells are programmed in the G1 phase

of the cell cycle to continue, or alternatively, to stop cell cycle progression In the latter case, the cells are induced to differentiate, undergo apoptosis or enter a quiescent or senescent state (Shackelford et al 1999)

The cyclin-dependent kinases (CDKs) are the main drivers of the progression through

the cell cycle In budding yeast Cdc28 is the sole essential Cdk (homologous to the mammalian Cdc2 or Cdk1) Cdc28 (Cdk1) is a highly conserved serine/threonine protein kinase whose activity is required for transition through the different phases of the cell cycle (Nasmyth 1993) Cdc28 is catalytically inactive and requires the binding of its regulatory partners, known as cyclins, for activity Cdc28 can associate with different types of cyclins with different Cdc28/cyclin complexes active at different periods during the cell cycle mediating specific cell cycle transitions (Figure1) At late G1, the passage through START (operationally defined as a small window after which cells are irreversibly committed to a new division cycle) requires activation of Cdc28 by G1 cyclins Cln1, Cln2 and Cln3 (Bloom et al 2007) This is also the time the daughter cell emerges as a small bud from the mother cell The bud

continues to grow throughout the cell cycle until it reaches almost the same size as the mother cell and separates from the mother during cytokinesis Soon after traversing START, association of Cdc28 with cyclins Clb5 and Clb6 promotes initiation of S phase during which DNA is replicated (Epstein et al 1992; Kuhne et al 1993; Schwob et al 1993; Basco et al 1995) Upon completion of DNA synthesis and a short G2 phase, Cdc28 forms a complex with mitotic cyclins (Clb1, 2, 3, 4) to facilitate progression into mitosis (M phase) Towards the end of M phase, cyclins are proteolytically degraded resulting in a rapid decrease in Cdc28 activity, thus allowing cells to exit mitosis Among the four different mitotic complexes, Cdc28/Clb2 contributes the highest mitotic kinase activity (Surana et al 1991; Fitch

et al 1992; Richardson et al 1992)

1.2.3 Regulation of Cdk activity

The activity of Cdk1 is regulated at diverse levels As discussed earlier, sequential expression of Cln and Clb cyclins impose control on the Cdk activity at the transcriptional level Cyclin association is insufficient to activate Cdc28 to its full capacity; a number of post-translational events such as phosphorylation and dephosphorylation are essential

Phosphorylation of a conserved Threonine-169 (similar to Thr-167 in S

pombe and Thr-161 in human) in the T-loop adjacent to the kinase active site is

catalyzed by the Cdk-activating kinase (CAK) (Gould et al 1991); (Kaldis et al 1996) This modification promotes stabilization of Cdc28/Clb complex In addition

to this, phosphorylation at the highly conserved Tyrosine-19 (equivalent to Tyr-15 in

other organisms) within the ATP-binding domain by Swe1 kinase (an ortholog of

human Wee1) inactivates Cdk1 activity and prevents entry into mitosis (Booher et al 1993) Tyr19 is also the target of regulation by both the replication checkpoint (Sorger et al 1992; Rhind et al 1998; Krishnan et al 2004) and the DNA damage checkpoint (Amon et al 1992) This inhibitory phosphorylation can be reversed by dephosphorylation of the same residue by the conserved tyrosine-phosphatase Mih1 (orthologue of human Cdc25) that activates Cdk activity at the onset of mitosis (Russell et al 1989; Nurse 1990; Amon et al 1992) Other regulators of the master kinase Cdk1, known as Cdk inhibitors have also been identified In budding yeast, Sic1, an inhibitor of Cdc28/Clb kinase inhibits both the S phase kinase Cdc28/Clb5/Clb6 in late G1 and the mitotic kinase in late telophase to facilitate exit from mitosis (Deshaies 1997; Mendenhall et al 1998) Besides this, Far1 also inactivates the Cdc28/Cln complex in the context of pheromone-mediated G1 arrest (Gartner et al 1998)

1.2.4 Regulation of cell cycle events by checkpoints

To ensure that cell cycle events are orchestrated in a precise and orderly manner, eukaryotic cells have evolved surveillance mechanisms, known as “checkpoints” that monitor and coordinate cell cycle progression Checkpoints ensure that if an event is interrupted or executed erroneously, transition to the subsequent phase is suspended transiently to allow time for repairs before resumption of the cell cycle (Hartwell et al 1989)

In budding yeast, four major checkpoint controls have been described and studied extensively: morphogenetic checkpoint, DNA replication checkpoint, DNA damage checkpoint, and spindle assembly checkpoint The morphogenetic

checkpoint monitors proper bud formation (Lew et al 1995) It delays cell cycle progression in response to to a defect in cell polarity that prevents bud emergence The replication checkpoint is triggered by stalled replication forks (for e.g., when DNA synthesis is interrupted by drugs such as hydroxyurea) and prevents the premature onset of mitosis until DNA replication is completed (Osborn et al 2002) The DNA damage checkpoint responds to any physical damage to the DNA such as DNA alkylation (caused by the DNA damaging drug, MMS [methylmethanesulfonate]), UV mediated cross linking and other genotoxic stresses (Melo and Toczyski 2002) Any perturbation in various aspects of spindle dynamics

or spindle assembly, absence of bi-orientation and lack of tension across sister kinetochores are monitored by the spindle assembly checkpoint Another less studied checkpoint - called the spindle positioning checkpoint - prevents mitotic exit and cytokinesis if spindles are misaligned with respect to the mother-bud axis (Lew et al 2003)

1.3 Regulation of cell cycle by protein degradation

Proteolytic destruction is a crucial determinant of virtually all biological processes including cell cycle progression from yeast to human The degradation systems play

an important role in the maintenance of cellular homeostasis by controlling the

stability of numerous regulators such as cell cycle proteins (e.g., cyclins, CDK

inhibitors and replication factors), transcription factors, tumour suppressor proteins,

membrane proteins and many more

1.3.1 The ubiquitin-proteasome system

The fact that the Nobel prize in chemistry 2004 was awarded jointly to Avram Hershko, Aaron Ciechanover and Irwin Rose for the discovery of ubiquitin-mediated protein degradation signifies its fundamental importance in the regulation of a wide range of cellular activities Generally proteins targeted for degradation by this pathway undergo two successive events: (i) covalent attachment of multimers of protein known as ubiquitin (a polypeptide of 76 amino acids) to the substrate protein

in a process known as ubiquitylation (Hershko et al 1998); (Hochstrasser 1996) and (ii) the ATP-dependent proteolysis of the substrate protein by a gigantic, multi-subunit protease assembly known as the proteasome (Pickart 2001) Ubiquitylation of

a substrate protein requires the activity of E1 activating), E2 conjugating) and E3 (ubiquitin ligase) enzymes The C-terminal glycine of ubiquitin (Gly76) is first bound via a high-energy thioester bond to a cysteine residue in the active site of E1 enzyme, in an ATP-coupled reaction Then, E2 enzyme transiently receives the activated ubiquitin from E1 enzyme, again as a thiolester linkage on a cysteine residue Finally, ubiquitin is transferred from E2 enzyme to a lysine side-chain on the substrate protein via an isopeptide bond This critical final step is mediated by an E3 enzyme Repeated transfer of additional ubiquitin moieties to successive lysines on each previously conjugated ubiquitin forms a polyubiquitin chain Usually a polyubiquitin chain comprising minimally of four ubiquitin monomers is sufficient for recognition by the 26S proteasome for degradation (Willems et al 2004) In some target proteins that lack lysine residues, polyubiquitylation can occur on the amino group at the N terminus of the substrate protein (Ciechanover et al 2004; Ciechanover et al 2004) The attachment of ubiquitin on different lysine residues can determine the fate of the protein

(ubiquitin-Polyubiquitin chains that are linked through Lys48 (well studied) and Lys29 (less studied) usually act as a signal for proteasome mediated degradation (Nandi et al 2006) Polyubiquitylation at Lys63 may act as a signal for DNA repair but not degradation (Weissman 2001) Moreover, monoubiquitination of proteins contribute

to other pathways such as endocytosis, histone regulation, virus budding and others (Hicke 2001)

In the cell cycle context, SCF (Skp1, Cullin, F box protein complex) and APC (Anaphase Promoting Complex) are the two main multi-subunit E3 ubiquitin ligases that play essential roles in G1-S, G2-M and M-G1 transitions

1.3.2 SCF

The name SCF was derived from three of its constituent components - Skp1, Cullin and F-box – which were discovered to be essential genes for cell cycle progression in budding yeast (Cardozo et al 2004) The invariant core complex of these SCF multisubunit enzymes is composed of the Skp1 linker protein, the Cdc53/Cul1 scaffold protein and the Rbx1/Roc1/Hrt1 RING domain protein (Patton et al 1998; Deshaies 1999; Tyers et al 2000) Subsequent studies demonstrated direct interactions between Skp1, Cdc53 and various F-box proteins; all were shown to be important in the recruitment of specific target proteins for ubiquitylation by an associated E2 enzyme (Cdc34) The different F-box sub-units determine substrate specificity and recruit substrates via their specific protein-protein interaction domains

known as WD-40 repeats (present in the CDC4 and MET30 genes) or the leucine-rich repeats (LRR; in the GRR1 gene) (Bai et al 1996) Budding yeast contains at least 21

known F-box proteins but some with unknown function (Willems et al 2004) A

brief description of some of the best-understood SCF-dependent regulatory mechanisms in the yeast cell cycle follows below

1.3.2.1 SCFCdc4

The initial insight into SCF-dependent proteolysis came from analysis of the S

cerevisiae cell division cycle (cdc) mutants cdc4, cdc34, cdc53 (Patton et al 1998)

At the non permissive temperature, these mutants arrest in G1 phase with unreplicated DNA and multiple elongated buds Additional genetic and biochemical studies revealed that the E3 ubiquitin ligase complex (comprising Cdc53, Skp1 and Rbx1) acts in synergy with F-box protein (Cdc4) and E2 enzyme Cdc34 to regulate G1/S transition (Schwob et al 1994; Mathias et al 1996; Verma et al 1997) Budding yeast SCFCdc4 targets important regulatory proteins such as CDK inhibitors Sic1 and Far1, the replication protein Cdc6 and transcription factor Gcn4 (Patton et al 1998)

Sic1 is a Cdc28/Clb inhibitor that is essential for the establishment of CDK-free

interval in G1 phase necessary for the assembly of pre-replicative origins of DNA replication (Schwob et al 1994) In late G1 phase, Cdc28-Cln1/Cln2/Cln3 kinase complexes phosphorylate Sic1 and target it to SCFCdc4 for SCF-dependent degradation This allows the activation of S-phase specific Cdc28/Clb5-Clb6 complexes triggering

entry into S phase Sic1 must be phosphorylated on at least six CDK-specific sites in order to bind Cdc4 At the end of mitosis, however, the anti-CDK phosphatase Cdc14

maintains the mitotically-expressed Sic1 in an unphosphorylated and stable form (Visintin et al 1998)

Another CDK inhibitor, Far1, is also targeted by SCFCdc4 via Cln1/Cln2/Cln3-dependent phosphorylation, in the absence of mating pheromone

Cdc28-(McKinney et al 1993) Upon pheromone stimulation, Far1 is activated via phosphorylation by MAPK Fus3 to inhibit Cdc28/Cln kinases leading to G1 phase arrest that allows cells to halt cell cycle and prepare for mating (Peter et al 1993; Tyers et al 1993)

Another SCFCdc4 substrate, Cdc6 plays an important role in the establishment

of pre-replicative complexes at the origins of replication in G1 cells (Diffley 1996)

At the onset of S phase, Cdc6 is degraded to prevent re-formation of the replicative complexes until the end of the division cycle This constitutes a part of the regulatory strategy that enforces on DNA replication the ‘once per cell cycle’ rule Cdc6 degradation requires both SCFCdc4 and CDK phosphorylation throughout the cell cycle (Drury et al 1997; Elsasser et al 1999; Drury et al 2000; Perkins et al 2001)

In late G1 and S phase, Cdc6 is primed for degradation via phosphorylation at its terminus by Cdc28/Cln complexes, whereas in G2-M phase, it is the C-terminus that

N-is phosphorylated by the CDK/Clb-complexes (Drury et al 2000; Perkins et al 2001) The alternate modes of phopshorylation suggest that binding of Cdc6 to SCFCdc4 may

be affected by conformational changes resulting from cell-cycle regulated Cln or Clb/Cdc6 interactions (Perkins et al 2001)

Gcn4 is another target-substrate of Cdc4 Gcn4 is a transcriptional activator that regulates amino acid biosynthesis genes and is degraded in non-starving conditions It is recognized by Cdc4 after phosphorylation by two cyclin-dependent kinases, Pcl5/pho85 and Srb10/Srb11 (Kornitzer et al 1994; Meimoun et al 2000; Chi et al 2001; Shemer et al 2002)

1.3.2.2 SCFGrr1

The G1 cyclins Cln1 and Cln2 are two primary, well-characterized substrates for

Cdc28-dependent autophosphorylation on multiple sites (Willems et al 1996) The recognition of phospho-Cln1 and phospho-Cln2 are dependent on the basic surface residues in the LRR-domain of Grr1 (Skowyra et al 1997; Patton et al 1998) However, the F-box protein for Cln3, another G1 cyclin, has not yet been identified (Tyers et al 1992)

In addition, SCFGrr1 also catalyzes ubiquitination of Gic2 protein at G1-S transition (Willems et al 2004) Gic2 is a downstream effector of Rho-related GTP-binding protein Cdc42, which is crucial for the initiation of polarization of the actin cytoskeleton during bud emergence The instability of Gic2 is also dependent on phosphorylation by unknown kinases before it is recognized and subjected to SCFGrr1-dependent proteolysis (Jaquenoud et al 1998) The phosphorylation of Gic2 only occurs after it is activated by Cdc42 (Jaquenoud et al 1998) This coupling of Gic2 activation and proteolysis restrict Gic2 activity to a narrow window in G1 phase

(Patton et al 2000)

1.3.3 APC

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase known to catalyze ubiquitylation reactions and contains more than a dozen subunits that assemble into a large 1.5-MDa complex (twelve subunits have been identified in humans and thirteen

in budding yeast) (Zachariae et al 1999; Harper et al 2002; Peters 2006) APC is also thought to be a distant relative of SCF as both contain subunits with cullin and RING-finger domains Similar to SCF, APC assembles polyubiquitin chains on substrate proteins and targets these proteins for destruction by 26S proteasome The ubiquitylation process requires APC in association with three other cofactors, the ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a co-activator protein As described earlier, just like all E3 enzymes, APC utilizes

ubiquitins that are activated by E1 and transfers them to E2 enzymes Both UBCH5 and UBCH10 (which is also known as E2-C or UbcX) are two commonly found E2

enzymes in human cells, Drosophila melanogaster and fission yeast (Aristarkhov et al

1996; Yu et al 1996) UBCH10 is essential for the initiation of anaphase (in chromosome segregation) (Townsley et al 1997; Seino et al 2003; Mathe et al 2004) However, Ubc5 alone in budding yeast seems to be sufficient for APC function (Townsley et al 1998)

In addition to E2 enzymes, APC activity is strictly dependent on one or several co-activator proteins that associate with APC at different stages of the cell cycle The best characterized co-activator proteins are Cdc20 and Cdh1 (Peters 2006) All of these proteins possess sequence elements, known as the N-terminal C-box (consensus sequence: DRF/YIPXR) and the C-terminal IR-tail that mediate binding to APC at its tetratrico peptide repeat (TPR) subunits, thus facilitating the ubiquitylation process (Schwab et al 2001; Passmore et al 2003; Vodermaier et al 2003)

1.3.3.1 Substrate specificity by APC

How are substrates recognized by APC? As described earlier, APC requires activator proteins such as Cdc20 or Cdh1 (also known as Hct1) for substrate ubiquitylation These co-activator proteins contain a C-terminal WD40 domain that folds into a propeller-like structure, which is believed to recognize APC substrates by interacting with specific elements in these substrates, called destruction or D-boxes (containing consensus amino acid sequence motif RXXLXXXN) and KEN-boxes (consensus motif KEN) (Glotzer et al 1991; Pfleger et al 2000) Other recognition-motifs such

as A-box and the GxEN motif have also been identified (Littlepage et al 2002; Castro

(Burton et al 2001; Hilioti et al 2001; Pfleger et al 2001) The above observations support the “substrate-adaptor” hypothesis where the WD40 domain of co-activator proteins recognize specific substrates through direct interaction with conserved motifs such as D and KEN boxes, and recruit them to APC for processive substrate ubiquitylation (Carroll et al 2002); (Kraft et al 2005) However, findings from Tim Hunt’s group cast doubt on the “substrate-adaptor” hypothesis They reported that cyclin B and the mitotic kinase Nek2A (substrates) can directly bind APC from Xenopus egg extracts in the absence of Cdc20 and Cdh1 (co-activators) (Yamano et al 2004)

The role of APC in substrate recognition is mysterious A very surprising discovery showed that both interactions between substrates and co-activators as well

as between substrates and APC are D-box dependent (Yamano et al 2004; Burton et

al 2005; Eytan et al 2006) The APC subunits known as Doc1 is postulated to mediate interactions between substrates and APC since APC lacking Doc1 exhibits processivity defect (Carroll et al 2002) Moreover, there is evidence that Doc1 contributes either directly or indirectly to D-box recognition by APC (Passmore et al 2003; Carroll et al 2005) It is undeniable that a greater understanding of the

structural interaction between Cdc20/Cdh1 and substrates containing the degradation motifs will be required to prove different hypotheses concerning substrate specificity and substrate ubiquitylation It is also essential that APC can selectively recognize substrates at the correct time for several key events in mitosis- the initiation of anaphase, exit from mitosis and the preparation for the next round of DNA replication The reason why APC-dependent degradation reactions must be tightly regulated is because inappropriate activation of APC can cause fatal coordination-errors in cell cycle progression Therefore, it is mandatory that Cdc20 and Cdh1 activate APC in a sequential manner in mitosis, with APCCdc20 activation at anaphase onset followed by APCCdh1 at the end of mitosis through G1

1.3.3.2 Regulation of anaphase by APC

The anaphase promoting complex (APC) was named for its best-known and most important function in the regulation of anaphase onset Upon completion of S phase, duplicated sister chromatids are glued together by the cohesin complex to promote biorientation and prevent premature chromosome segregation (Nasmyth 2002) Securin (Pds1) also binds to the protease, separase (Esp1), and causes its inhibition to ensure that the cohesin complex remains intact In late metaphase, when all kinetochores assembled at the centromeric region of duplicated sister chromatids are attached to microtubules emanating from opposite poles, APC is activated by Cdc20 (Peters 2006) At the onset of anaphase, APCCdc20 targets Pds1 for ubiquitin-mediated destruction, thus liberating Esp1 (yeast separase) The activated, free Esp1 cleaves the Scc1 subunit of cohesin complex, thus dissolving the cohesion between sister chromatids to allow partitioning of sister chromatids by the mitotic spindle (Uhlmann