THE REPLICATION OF THE TWO HOTSPOTS OF BREAKAGE
LOCATED WITHIN THE HUMAN COMMON FRAGILE SITE FRA11D
OCCURS IN MID TO LATE S PHASE: A PRELIMINARY STUDY
By
Omar El Mawas
A thesis submitted to the Department of Biology in partial fulfillment of the requirements for
the degree of Master of Science in Biology
Faculty of Sciences
University of Balamand
December 2015
Copyright © 2015, Omar El Mawas
All Rights Reserved
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University of Balamand
Faculty of Sciences
This is to certify that I have examined this copy of a Master’s thesis by
Omar El Mawas
and have found that it is complete and satisfactory in all the respects,
and that any and all revisions required by the final
examining jury have been made.
JURY MEMBERS:
Approved: ____________________
Full name, Ph.D.
President of the jury
Approved: ____________________
Full name, Ph.D.
Jury Member
Approved: ____________________
Full name, Ph.D.
Supervisor
Date of thesis defense: December 16, 2015
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ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my advisor Dr. .…… for his continuous
support, for his patience, motivation, enthusiasm, and immense knowledge. His guidance
helped me in all the time of research and writing of this thesis.
I also would like to thank my family for supporting me spiritually throughout my life.
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ABSTRACT
Cancer is a genetic disease characterized by the transformation of normal cells into
malignant cells through uncontrollable divisions. Genomic instability, a key feature of
genomic regions called fragile sites (FS), has been shown to be a hallmark of cancer. FS are
specific DNA loci that show propensity to form gaps and breaks on metaphase chromosomes
following DNA replication stress…. Calyculin A, which triggers premature chromosome
condensation at any phase of the cell cycle, also induces CFS. The causes of their fragility are
not yet fully deciphered; several causes are so far described such as replication fork stalling,
paucity in initiation events, and late or slow replication. This latency might be due to CFS
forming secondary structures, which upset the progression of the replication fork. In our
work, we propose to investigate the replication timing at the CFS FRA11D which is located
within the chromosomal band 11p14.2. …
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS
iii
ABSTRACT
iv
TABLE OF CONTENTS
v
LIST OF ABBREVIATIONS
viii
LIST OF TABLES
x
LIST OF FIGURES
xi
CHAPTER 1: INTRODUCTION
1
1.1 The Cell and Cell Cycle
1
1.1.1 Cell Discovery
1
1.1.2 Cell Cycle: Description and Regulation
2
1.2 Cancer
1.2.1 Cancer Hallmarks
6
7
1.2.1.1 Limitless replication potential
7
1.2.1.2 Sustaining proliferative potential
8
1.2.1.3 Insensitivity to antigrowth signals
8
1.2.2 Proto-Oncogenes and Tumor Suppressor Genes
12
1.2.3 Cancer Risk Factors
13
1.2.3.1 Genetic predisposition
1.2.4 Cancer Sign and Symptoms
13
16
1.2.4.1 Unexpected weight loss
16
1.3.6.2.5 Inability to recover stalled forks
39
1.4 Calyculin A
40
1.5 Fluorescent in Situ Hybridization
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CHAPTER 2: MATERIALS AND METHODS
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2.1 Lymphocyte Culture and CFS Induction
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2.2 Metaphase Spreading
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CHAPTER 3: RESULTS
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3.1 Lymphocyte Culture: Induction of Premature Chromosome Condensation and
Metaphase Spreading
54
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3.2 Bacterial Culture
57
CHAPTER 4: DISCUSSION
95
LIST OF REFERENCES
100
APPENDIX A: Gene Maps
105
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LIST OF ABBREVIATIONS
AML
Acute Myelogenous Leukemia
APC
Anaphase-Promoting Complex
APH
Aphidicolin
AT
Ataxia Telangiectasia
ATM
Ataxia Telangiectasia Mutated
ATR
Ataxia Telangiectasia and Rad3-Related Protein
BAC
Bacterial Artificial Chromosome
BRCA1
Breast Cancer 1
BrdU
Bromodeoxyuridine
CDK
Cyclin-Dependent Kinase
CFS
Common Fragile Sites
DM
Double Minutes
dsDNA
Double Stranded DNA
FHIT
Fragile Histidine Triad
FISH
Fluorescent In Situ Hybridization
FMR1
Fragile Mental Retardation 1
FMR2
Fragile Mental Retardation 2
FS
Fragile Sites
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LIST OF TABLES
Table 1.1
CACGs and Molecularly Mapped CFS Involved in Cancer.
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Table 2.1
Standard PCR Protocol.
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Table 3.1
Forward and Reverse Primers Used for the Amplification of our Target
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Sequences.
Table 3.2
Classification of FISH Signals at the Level of All Clones According to
Mitotic Stages and Signal Shape.
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LIST OF FIGURES
Figure 1.1
The Stages of the Cell Cycle.
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Figure 1.2
APC Targeting Securing and Mitotic Cyclin for Degradation
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Allowing for Cell Cycle Progression.
Figure 1.3
The Cell Goes Through Several Stages Before It Reaches Metastasis
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and Becomes Malignant.
Figure 1.4
Cancer Cells Detach From the Primary Tumor, Squeeze through
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Blood Vessels and Form Secondary Tumors.
Figure 1.5
Acquired Capabilities in Parallel Pathways, Regardless of their
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Chronological Order, Eventually Lead to Colorectal Cancer.
Figure 1.6
Chromosome Ideogram Depicting the Locations of Frequently (Red)
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and Less Frequently (Blue) Expressed CFS.
Figure 1.7
Repeat Copy Number Increase as Suggested by Okazaki Fragment-
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Mediated Model.
Figure 1.8
Broad Scheme Depicting Possible Sources of Replication Stress at
CFS.
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1
CHAPTER 1
INTRODUCTION
1.1 The Cell and Cell Cycle
1.1.1 Cell Discovery
The mid-17th century witnessed one of the possibly greatest scientific breakthroughs,
the discovery of the cell. Thanks to the advances in microscopy, English physicist Robert
Hooke was able to examine thin slices of cork in 1665. He noticed that such tissues are
formed by tiny pores, he termed cells from the Latin word Cella meaning 'a small room'. …
Further studies into the cell led Walther Flemming to the discovery that cells undergo cellular
division or Karyomitosis (meaning threadlike metamorphosis of the nucleus) and his work
was published in 1882. Cell division is required both in developmental and adult stages to
maintain homeostasis. In the adult stage, it replaces old, warn out cells with new functional
ones (Mazzarello, 1999).
1.1.2 Cell Cycle: Description
The order of events required through which a cell can pass from one cell division to the
next is termed cell cycle. Today, we know that the cell cycle consists of two successive
stages: interphase, a highly regulated long preparatory stage, and mitosis, a unidirectional and
tightly regulated process by which one parent cell divides to give rise to two identical
daughter cells. In most mammalian cells, Mitosis, lasting one hour, includes prophase,
metaphase, anaphase and telophase. While interphase, lasting around twenty-three hours,
includes G1, S and G2 phases. G1 is a preparatory growth stage for DNA replication and
division, S is the stage where replication takes place, and G2 is the stage where the cell
prepares for mitosis (Vermeulen, Van Bockstaele, & Berneman, 2003).
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1.1.3 Cell Cycle Regulation: Cyclin – Cyclin Dependent Kinases
Most cells in an adult organism are not dividing. They remain inactive in a quiescent
stage called G0. Yet still each second twenty five million cells are undergoing cellular
division in an adult human. Such an enormous number demands an accurate regulation. As
such, specific cyclin proteins and cyclin-dependent kinases (CDKs) coordinately control the
progression of the cell from one stage to the next. CDKs are serine/threonine kinases.
Unbound CDKs are present in an inactive conformation (Collins & Garrett, 2005). The
association of cyclin proteins to CDKs triggers conformational changes in CDKs allowing
them to become catalytically active. It is noteworthy that the levels of CDKs in the cell
remain moderately constant throughout the cell cycle. Apart from cyclin D levels, which
increases in the beginning of G1 and remains constant throughout, the levels of cyclin
fluctuate while the cell progresses through its cycle. Cyclins are classified according to the
stage where they are mostly elevated. The level of cyclin in the cell is determined by the rate
of synthesis, transcriptional regulation of the cyclin gene, and protein degradation by
proteasome (Pecorino, 2012). The following Figure 1.1 highlights the different stages of the
cell cycle.
Figure 1.1: The Stages of the Cell Cycle (Pecorino, 2012).
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Growth factors such as c-MYC and c-fos induce the cell to leave G0 and re-enter the
cell cycle by inducing the expression of cyclin D and its partner CDK4/6. The interaction of
cyclin D with CDKs 4/6 leads the cell through G1…. Cyclin E interacts with CDK2 and
promotes the hyperphosphorylation of Rb by means of a positive-feedback loop. As a result,
a conformational change takes place in the pocket domain of Rb causing the release of the
E2F. E2F target genes (cyclin A, thymidylate synthase, etc.) become fully expressed and the
cell can proceed through the S phase (Haering, Lowe, Hochwagen, & Nasmyth, 2002).
…Other CFS that have been shown to be associated with cancer include FRA7G which
is located at the band 7q31.2 and has been shown to be frequently expressed in prostate,
breast, and ovarian cancer (Huang, et al., 1998). Central deletions in FRA6E, where the
candidate tumor suppressor gene Parkin lies, have been shown to exist in ovarian and lung
cancers. The following Table 1.1 shows molecularly mapped CFS and Cancer associated CFS
Genes (CACGs).
Table 1.1: CACGs and Molecularly Mapped CFS Involved in Cancer.
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1.3.6 Mechanism of Instability at FS
1.3.6.1 Mechanism of instability at RFS
The replication slippage model proposed by Sutherland et al. suggests that the Okazaki
fragment plays an important role during the mechanism of expansion of microsatellite CCG
or AT-rich minisatellites. In one case where the number of copies of repeats is less than 80, it
suggests that slippage of Okazaki fragment might still occur during polymerization though
the 5’ end of the Okazaki fragment might be firmly anchored by a unique sequence of DNA
at one side….
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LIST OF REFERENCES
Arabshahi, L., Brown, N., Khan, N., & Wright, G. (1988). Inhibition of DNA polymerase
alpha by aphidicolin derivatives. Nucleic Acids Research, 16(11), 5107-5113.
Baynton, K., Otterlei, M., Bjoras, M., von Kobbe, C., Bohr, V. A., & Seeberg, E. (2003).
WRN interacts physically and functionally with the recombination mediator protein
RAD52. Journal of Biological Chemistry, 278(38), 36476-36486. doi:
10.1074/jbc.M303885200
Berchuck, A., Heron, K. A., Carney, M. E., Lancaster, J. M., Fraser, E. G., Vinson, V. L., &
Frank, T. S. (1998). Frequency of germline and somatic BRCA1 mutations in ovarian
cancer. Clinical Cancer Research, 4(10), 2433-2437.
Bianchini, F., Elmstahl, S., Martinez-Garcia, C., van Kappel, A. L., Douki, T., Cadet, J., ...
Kaaks, R. (2000). Oxidative DNA damage in human lymphocytes: Correlations with
plasma levels of alpha-tocopherol and carotenoids. Carcinogenesis, 21(2), 321-324.
Cheng, C. H., & Kuchta, R. D. (1993). DNA polymerase epsilon: Aphidicolin inhibition and
the relationship between polymerase and exonuclease activity. Biochemistry, 32(33),
8568-8574.
Cliby, W. A., Roberts, C. J., Cimprich, K. A., Stringer, C. M., Lamb, J. R., Schreiber, S. L.,
& Friend, S. H. (1998). Overexpression of a kinase-inactive ATR protein causes
sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO
Journal, 17(1), 159-169. doi: 10.1093/emboj/17.1.159
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APPENDIX A: Gene Maps
Gene maps of the chloroplast transformation vectors. The egfp sequence, coding for
enhanced green fluorescent protein, with the respective psbA promoters and rrnB T1
terminator were cloned into MSC of pBluescript SK+ cloning vector.
Figure A.1: Transformation Vector for P. sativum, Final Size 4 165 bp
Figure A.2: Transformation Vector for V. litorea, Final Size 4 876 bp