The Replication of the Two Hotspots of Breakage Located Within the Human Common Fragile

1

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

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

examiningjury 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

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 familyfor supporting me spiritually throughout my life.

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. …

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
1.1The Cell and Cell Cycle / 1
1.1.1 Cell Discovery / 1
1.1.2 Cell Cycle: Description and Regulation / 2
1.2 Cancer / 6
1.2.1 Cancer Hallmarks / 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 / 13
1.2.4 Cancer Sign and Symptoms / 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 / 42
CHAPTER 2: MATERIALS AND METHODS / 49
2.1 Lymphocyte Culture and CFS Induction / 49
2.2 Metaphase Spreading / 49
CHAPTER 3: RESULTS / 54
3.1 Lymphocyte Culture: Induction of Premature Chromosome Condensation and Metaphase Spreading / 54
3.2 Bacterial Culture / 57
CHAPTER 4: DISCUSSION / 95
LIST OF REFERENCES / 100
APPENDIX A: Gene Maps / 105

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

LIST OF TABLES

Table 1.1 / CACGs and Molecularly Mapped CFS Involved in Cancer. / 31
Table 2.1 / Standard PCR Protocol. / 50
Table 3.1 / Forward and Reverse Primers Used for the Amplification of our Target Sequences. / 59
Table 3.2 / Classification of FISH Signals at the Level of All Clones According to Mitotic Stages and Signal Shape. / 66

LIST OF FIGURES

Figure 1.1 / The Stages of the Cell Cycle. / 3
Figure 1.2 / APC Targeting Securing and Mitotic Cyclin for Degradation Allowing for Cell Cycle Progression. / 4
Figure 1.3 / The Cell Goes Through Several Stages Before It Reaches Metastasis and Becomes Malignant. / 7
Figure 1.4 / Cancer Cells Detach From the Primary Tumor, Squeeze through Blood Vessels and Form Secondary Tumors. / 11
Figure 1.5 / Acquired Capabilities in Parallel Pathways, Regardless of their Chronological Order, Eventually Lead to Colorectal Cancer. / 12
Figure 1.6 / Chromosome Ideogram Depicting the Locations of Frequently (Red) and Less Frequently (Blue) Expressed CFS. / 25
Figure 1.7 / Repeat Copy Number Increase as Suggested by Okazaki Fragment-Mediated Model. / 32
Figure 1.8 / Broad Scheme Depicting Possible Sources of Replication Stress at CFS. / 34

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).

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).

Growth factors such as c-MYCand 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 (cyclinA, 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.

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….

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

APPENDIX A: Gene Maps

Gene maps of the chloroplast transformation vectors. The egfpsequence, coding for enhanced green fluorescent protein, with the respective psbApromoters and rrnBT1 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