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«A Dissertation Presented to The Academic Faculty By Hyun-Min Kim In partial fulfillment of the requirements for the degree Doctor of Philosophy in ...»

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Genome instability induced by triplex forming mirror repeats in S.cerevisiae

A Dissertation

Presented to

The Academic Faculty

By

Hyun-Min Kim

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy in the School of Biology

Georgia Institute of Technology

May 2009

Copyright 2009 by Hyun-Min Kim

Genome instability induced by triplex forming mirror repeats in S.cerevisiae

Approved by:

Dr. Kirill Lobachev Dr. Nicholas Hud Academic advisor School of Chemistry and Biochemistry School of Biology Georgia Institute of Technology Georgia Institute of Technology Dr. Francesca Storici Dr. Yury Chernoff School of Biology School of Biology Georgia Institute of Technology Georgia Institute of Technology Dr. SooJin Yi School of Biology Georgia Institute of Technology Date approved; March 20 2009 Acknowledgements I would like to extend my earnest appreciation to my academic advisor Dr. Kirill Lobachev for his scientific guidance and ambitious motivation during my PH.D program.

I would like to thank Dr. Natasha Degtyareva for her help with the scientific guide and critical review.

I would like to thank my thesis committee members Dr. Nicholas Hud, Dr. Soojin Yi, Dr.

Yuri Chernoff, and Dr. Francesca Storici.

I would like to thank Katy Bruce for the proofreading and Gary Newnam for his help.

I would like to thank all the undergraduate students, especially Shadeah Suleiman and George Laskar.

I would like to thank my parents Soo-Woong Kim and Cha-Soon Hwang and my brother Hyun-Dong Kim for their unconditional supports.

iii Table of Contents ACKNOWLEDGEMENTS 

TABLE OF CONTENTS 

.

LIST OF FIGURES 

LIST OF TABLES 

  CHAPTER I. BACKGROUND AND SIGNIFICANCE 

1.1. REPETITIVE SEQUENCES THAT CAN ADOPT SECONDARY STRUCTURES INDUCE GENOME INSTABILITY

1.2. TRIPLET REPEATS EXPANSION OCCURRENCES AND CONSEQUENCES 

1.2.1. TRIPLET REPEATS OCCUR NATURALLY IN THE HUMAN GENOME AND EXPANDED TRACKS LEAD TO 

NEURODEGENERATIVE DISEASES 

FIGURE 1. LOCATIONS OF EXPANDABLE TRINUCLEOTIDE REPEATS RESPONSIBLE FOR HUMAN DISEASES.

1.2.2. EXPANDED TRIPLET REPEATS AFFECT GENE EXPRESSION AND/OR PROTEIN FUNCTION. ............... 4 

1.3. LONG TRACKS OF TRIPLET REPEATS (CAG/CTG AND CCG/CGG) INDUCE DOUBLE STRANDED BREAKS 

AND CHROMOSOME REARRANGEMENTS. 

1.4. INDIRECT EVIDENCE SUGGESTS GAA/TTC EXPANSION ‐ INDUCED CHROMOSOME FRAGILITY. ......... 5 

FIGURE 2.    EXAMPLES OF NON‐CANONICAL DNA SECONDARY STRUCTURES ADOPTED BY REPETITIVE 

SEQUENCES 

–  –  –

DNA’: PU PU PY AND PY PU PY 

–  –  –

FIGURE 8. MODEL TO SHOW HOW AZACYANINE AUGMENTED TRIPLEX‐MEDIATED FRAGILITY LEADS TO 

CELL DEATH. 

4.3. DISCUSSION 

FIGURE 9. AZACYANINE5 INDUCES SIZE VARIATION OF GAA/TTC REPEATS. 

FIGURE 10. AZACYANINES ARE NOT MUTAGENS 

4.4. REFERENCES 

4.5. MATERIALS AND METHODS 

4.5.1. STRAINS 

4.5.2. GENETIC TECHNIQUES 

4.5.3. 2D ANALYSIS OF REPLICATION FORK INTERMEDIATES 

TABLE 1. AZACYANINE AUGMENT CHROMOSOMAL ARM LOSS INDUCED BY (GAA) AND (TTC) REPEAT 

ORIENTATIONS. 

TABLE 2. AZACYANINES INHIBIT CELL GROWTH AND AUGMENT GENOME FRAGILITY IN A DOSE‐

DEPENDENT MANNER. 

TABLE 3. AZACYANINE 5 TREATMENT TRIGGERS SIZE VARIATION OF (GAA)230 REPEAT TRACKS.

 .......... 117    CHAPTER V. OVERALL CONCLUSIONS 

5.1. OVERALL CONCLUSIONS 

PUBLISHED OR IN PRESS 

–  –  –

CHAPTER I   

FIGURE 1. LOCATIONS OF EXPANDABLE TRINUCLEOTIDE REPEATS RESPONSIBLE FOR HUMAN DISEASES.

FIGURE 2.    EXAMPLES OF NON‐CANONICAL DNA SECONDARY STRUCTURES ADOPTED BY REPETITIVE 

SEQUENCES 

–  –  –

WILD TYPE CELLS WERE ARRESTED IN G2/M PHASE UPON TREATMENT WITH 0.2MM AZACYANINE 5.   

CELLS WERE IMAGED FOLLOWING PROPAGATION IN LIQUID SYNTHETIC COMPLETE MEDIUM WITH AND 

WITHOUT THE DRUG. OVER 60% OF THE CELLS WERE ARRESTED WITH LARGE BUDS FOLLOWING THE 

INCUBATION WITH THE AZACYANINE 5. TREATMENT WITH AZACYANINE 3 AND 5 YIELDED SIMILAR 

–  –  –

FIGURE 8. MODEL TO SHOW HOW AZACYANINE AUGMENTED TRIPLEX‐MEDIATED FRAGILITY LEADS TO 

CELL DEATH. 

FIGURE 9. AZACYANINE5 INDUCES SIZE VARIATION OF GAA/TTC REPEATS. 





FIGURE 10. AZACYANINES ARE NOT MUTAGENS 

–  –  –

TABLE 1. AZACYANINE AUGMENT CHROMOSOMAL ARM LOSS INDUCED BY (GAA) AND (TTC) REPEAT 

ORIENTATIONS. 

TABLE 2. AZACYANINES INHIBIT CELL GROWTH AND AUGMENT GENOME FRAGILITY IN A DOSE‐

DEPENDENT MANNER. 

TABLE 3. AZACYANINE 5 TREATMENT TRIGGERS SIZE VARIATION OF (GAA)230 REPEAT TRACKS.

 .......... 117 

–  –  –

1.1. Repetitive sequences that can adopt secondary structures induce genome instability Many cancers and hereditary diseases are characterized by chromosomal anomalies such as deletions, inversions, translocations and gene amplifications (Duker, 2002; Lengauer et al, 1998). Chromosomal aberrations can be a consequence of cell exposure to exogenous factors that cause DNA damage (e.g. ionizing radiation), or can be a result of malfunctioning of the endogenous systems, for example those involved in DNA metabolism.

Recent studies reveal that certain DNA motifs can also pose a threat to genome stability (Gordenin & Resnick, 1998; Lobachev et al, 2000).Unstable repetitive sequences such as triplet repeats, inverted repeats, and AT- and GC-rich micro- and minisatellites known as “at-risk motifs” (ARMs) promote genome rearrangements (Gordenin & Resnick, 1998).

Their ability to induce genome instability strongly depends on their potential to adopt non-canonical secondary structures (Callahan et al, 2003; Lobachev et al, 2000), Figure 1). Hairpin and cruciform structures can be formed by intra strand base pairing in one and in both DNA strands respectively. Because of internal symmetry of the inverted, AT, CG di-nucleotides and CTG/CAG, CGG/CCG triplet repeats these sequences could form hairpins and cruciforms (Sinden, 1994). In addition, some triplet repeats adopt intramolecular triplex DNA (GAA/TTC and CGG/CCG) or quadruplex DNA (CGG/CCG) structures via formation of Hoogsteen base-pairs between single or double

–  –  –

structures directly correlates with the size of repeats or repeat tracks (Mitas, 1997;

Pearson et al, 1998; Sutherland et al, 1998). Among the above mentioned repetitive sequences, triplet repeats are under heavy scrutiny owing to their known ability to expand and consequently affect human health.

1.2. Triplet repeats expansion occurrences and consequences 1.2.1. Triplet repeats occur naturally in the human genome and expanded tracks lead to neurodegenerative diseases Trinucleotide repeats can be found in both coding and non coding regions of the human genome. Expansions of CTG/CAG, CCG/CGG or GAA/TTC repeats are associated with nearly 25 human neuromuscular diseases and the list continues to grow (Table. 1) (Mirkin, 2007), (Pearson et al, 1998). The normal (non-disease) repeat size ranges from 5 to ~45 for CTG/CAG, 7 to ~60 for CCG/CGG and 7 to ~33 for GAA/TTC. Individuals with critical threshold length defined as “premutation size” of triplet repeats are more prone for expansions. For example, in individuals suffering from Friedreich’s ataxia, a common inherited ataxia, expanded GAA/TTC repeats of up to 1700 triplets can be found in intron 1 of the FRDA gene (Figure 1).

–  –  –

BPES, blepharophimosis and epicanthus inversus; CCD, cleidocranial dysplasia; CCHS, congenital central hypoventilation syndrome; DM, myotonic dystrophy; DRPLA, dentatorubral–pallidoluysian atrophy; FRAXA, fragile X syndrome; FRAXE, fragile X mental retardation associated with FRAXE site; FRDA, Friedreich’s ataxia; FXTAS, fragile X tremor and ataxia syndrome; HD, Huntington’s disease;HDL2, Huntington’sdisease-like 2; HFG, hand–foot–genital syndrome;HPE5, holoprosencephaly 5; ISSX, X-linked infantile spasm syndrome; MRGH, mental retardation with isolated growth hormone deficiency; OPMD, oculopharyngeal muscular dystrophy; SBMA, spinaland bulbar muscular atrophy; SCA, spinocerebellar ataxia;SPD, synpolydactyly.

–  –  –

Expansion of triplet repeats found in non-coding regions of genes can result in loss of protein function. For instance, expansion of CGG repeats (more than 200 repeats) in the 5’ UTR of the fragile X mental retardation 1 gene (FMR1) results in transcriptional silencing of the gene and loss of protein, causing alterations in dendritic functions in the diseased individuals. Polyglutamine diseases represent altered protein function disorders where expansion of triplet repeats in the coding regions of the gene leads to aggregation of the protein products. An example of such disorder is Huntington’s disease, which is caused by the expansion of CAG repeats in exon1 of the corresponding gene. Expanded triplet repeats can also lead to altered RNA function, as evidenced in diseases such as myotonic dystrophy, which is caused by expansion of CAG repeats in the 3’ of untranslated region.

1.3. Long tracks of triplet repeats (CAG/CTG and CCG/CGG) induce double stranded breaks and chromosome rearrangements.

Triplet repeat expansions can pose a threat to human health via adversely affecting gene/protein function or by affecting chromosome stability. Expanded triplet repeats act as fragile sites in vitro as revealed by constrictions in metaphase chromosomes of cells exposed to replication inhibitors (Nelson, 1995; Sutherland et al, 1998). Evidence for fragile site-mediated chromosome breakage in humans was first demonstrated in studies

–  –  –

patients with the syndrome coincide with the location of the expanded repeats. Also, expanded tracks of CCG/CGG and CTG/CAG repeats accentuate chromosome breakage not only at the site of the repeats expansion but also in the vicinity of the location of the repeats in yeast chromosomes (Balakumaran et al, 2000; Sutherland et al, 1998).

Number of observations supports an idea that the abnormal secondary structure of the triplet repeats act as inducer for chromosome instability. Both CAG/CTG and CCG/CGG trinucleotide repeats are prone to adopt non-canonical secondary structures such as hairpin DNA, cruciform structure, slipped strand structure and/or quadruplex (Figure 2). These expanded trinucleotide repeat tracts can cause replication arrest in vivo in yeast (Pelletier et al, 2003).

1.4. Indirect evidence suggests GAA/TTC expansion - induced chromosome fragility.

GAA/TTC trinucleotide repeats (TNR) are prone to adopt non-canonical secondary structures which are considered to be inducers of genome instability. For instance, the expanded GAA/TTC tracts can adopt abnormal secondary structures such as triplex DNA, sticky DNA, and/or hairpin DNA which could interfere with DNA replication, transcription, and/or recombination. Although GAA/TTC repeats have strong potential for inducing genome instability, their ability to induce chromosome fragility has never

–  –  –

plasmid. Often replication arrest can be converted to chromosome break which triggers chromosome rearrangements. As genome rearrangements are hallmark for cancer and human diseases, it is very important to understand the mechanism of chromosome fragility induced by GAA/TTC repeats. Moreover, it has been demonstrated that GAA/TTC repeats are highly abundant in the human genome (Clark et al, 2006; Clark et al, 2004; Clark et al, 2007), therefore they have high potential for induction of chromosome instability in multiple genomic locations which amplifies the significance of studying chromosome fragility potential caused by GAA/TTC repeats.

1.4.1. Expanded GAA/TTC repeats can adopt non-canonical secondary structure ‘triplex DNA’: Pu Pu Py and Py Pu Py Human genetics and model organism studies leave no doubts that non-canonical secondary structures, such as hairpin or cruciform DNA, triplex DNA, and quadruplex are strong inducers for chromosome instability (Wells, 2008). Since expanded tracts of GAA/TTC repeats are prone adaptation of non-canonical secondary structure such as triplex DNA, sticky DNA and hairpin DNA we hypothesize that these abnormal secondary structures can act as inducers of genome instability. Dependence of the level of transcriptional repression of frataxin and the extent of genomic instability in patients’

–  –  –



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