A study on premature segregation of unreplicated chromosomes 1

A STUDY ON PREMATURE SEGREGATION OF
UNREPLICATED CHROMOSOMES






KHONG JENN HUI













INSTITUTE OF MOLECULAR AND CELL
BIOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

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
i
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.
ii
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 SCF
Cdc4
…………… ……………………………………… 11
1.3.2.2 SCF
Grr1
……………………………………………………… 13
1.3.2.3 SCF
Met30
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
iii
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
iv
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
v
Bibliography…… …………………………………………………………… 166

ACKNOWLEDGEMENTS I!
TABLE OF CONTENTS II!
SUMMARY VI!
LIST OF TABLES IX!
LIST OF FIGURES X!
CHAPTER 1! INTRODUCTION 14!
1.1! Introductory Remarks 14!
1.2! Brief overview of cell cycle 15!
1.2.1! Saccharomyces cerevisiae cell cycle 15!
vi
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
vii
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
APC
Cdh1

and 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
viii
that initiation of DNA replication saves the cells from potential chromosome-
segregation catastrophe.



ix
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

x
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
APC
Cdc20
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
xi
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

xii
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
xiii
List of Symbols

Ab
Antibody
Bp
Base pairs
BSA
CDK
Bovine serum albumin
Cyclin Dependent Kinase
DAPI
4', 6-diamidino-2-phenylindole
DNA
Deoxyribonucleic acid
DIC
Differential interference contrast
DNA
Deoxyribonucleic acid
DTT
Dithiothreitol
EDTA
Ethylenediamine tetraacetic acid
g
Gram
Gal
Galactose

GFP
Green Fluorescent Protein
Glu
Glucose
h / hr
Hour
HA
Haemagglutinin
HR
Homologous Recombination
HRP
Horseradish peroxidase
IP
Immunoprecipitation
Kb
Kilobases
kDA
KiloDalton
M
Molar
MAPs
Microtubule associated proteins
Met
Methionine
mg
Milligram
µg
Microgram
mins
Minutes

ml
Milliliter
µl
Microliter
mM
Millimolar
mRNA
Messenger RNA
o
C
Degree Celsius
OD
Optical density
PBS
Phosphate-buffered saline
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


14
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
15
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
16
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.

17






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.

18
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 G1-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
19
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
20
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
21
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.

22
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 (ubiquitin-activating), E2 (ubiquitin-
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.
23
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