Gene-specific trinucleotide repeat expansions are the cause of an ever-growing number of disorders, including myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 7 (SCA7). Both DM1, and SCA7 are characterized by large differences in repeat numbers between tissues that are differentially affected, indicating tissue-specific mechanisms of repeat instability. The mechanism(s) of both somatic as well as germline instability are complex and still poorly understood, with evidence supporting the contribution of cis-elements, trans factors, and DNA metabolic processes that are hypothesized to involve alternative structure formation within the DNA tract. This thesis involves investigations into the role of a particular cis-element (CTCF) on instability, as well as the detection of slipped-DNAs in patient tissues and the presence of rare mutations within those same tissues. Here I identify the first endogenous cis-element reported to show regulation of instability at a trinucleotide repeat disease locus, the DNA binding site for the insulator protein CCCTC- binding factor (CTCF) downstream of the SCA7 repeat. Using a mouse model with a mutation in the CTCF binding domain, I show that the loss of CTCF binding stimulates germline and somatic instability in a tissue-specific and age-dependent manner. The binding of CTCF likely protects the repeat tract from expansion by shielding it from other elements that may contribute to expansion. DNA metabolic processes such as replication, repair, and transcription likely play a role in repeat expansion at disease loci, with the general mechanism hypothesized to be the extrusion and aberrant repair of slipped-DNA structures during the unwinding process for each. While characterizing DM1 patient tissues in order to isolate slipped-DNA structures, I characterized two non-CTG repeat insertion mutations that had completely replaced the repeat tract in a small subset of cells in only two tissues in one patient. Given the hypermutable nature of expanded repeat tracts, it is possible that these types of mutations are more common than suspected. Finally, I report on the detection and isolation of slipped-DNA structures from the endogenous DM1 locus from patient tissues. The slip-outs appear as clusters along a length of DNA, rather than single isolated slip-outs, and more unstable tissues contain greater amounts of slipped-DNA compared to more stable tissues. This detection implies that slipped-DNA structures are not merely transient intermediates in the mutation and expansion process as has long been assumed, but remain within the DNA at detectable levels. The data reported herein both furthers our understanding of trinucleotide repeat instability, and additionally confirms the decades-long hypothesis that slipped-DNAs are in fact forming in patient tissues in a tissue-specific manner.

Gene-specific trinucleotide repeat expansions are the cause of an ever-growing number of disorders, including myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 7 (SCA7). Both DM1, and SCA7 are characterized by large differences in repeat numbers between tissues that are differentially affected, indicating tissue-specific mechanisms of repeat instability. The mechanism(s) of both somatic as well as germline instability are complex and still poorly understood, with evidence supporting the contribution of cis-elements, trans factors, and DNA metabolic processes that are hypothesized to involve alternative structure formation within the DNA tract. This thesis involves investigations into the role of a particular cis-element (CTCF) on instability, as well as the detection of slipped-DNAs in patient tissues and the presence of rare mutations within those same tissues. Here I identify the first endogenous cis-element reported to show regulation of instability at a trinucleotide repeat disease locus, the DNA binding site for the insulator protein CCCTC- binding factor (CTCF) downstream of the SCA7 repeat. Using a mouse model with a mutation in the CTCF binding domain, I show that the loss of CTCF binding stimulates germline and somatic instability in a tissue-specific and age-dependent manner. The binding of CTCF likely protects the repeat tract from expansion by shielding it from other elements that may contribute to expansion. DNA metabolic processes such as replication, repair, and transcription likely play a role in repeat expansion at disease loci, with the general mechanism hypothesized to be the extrusion and aberrant repair of slipped-DNA structures during the unwinding process for each. While characterizing DM1 patient tissues in order to isolate slipped-DNA structures, I characterized two non-CTG repeat insertion mutations that had completely replaced the repeat tract in a small subset of cells in only two tissues in one patient. Given the hypermutable nature of expanded repeat tracts, it is possible that these types of mutations are more common than suspected. Finally, I report on the detection and isolation of slipped-DNA structures from the endogenous DM1 locus from patient tissues. The slip-outs appear as clusters along a length of DNA, rather than single isolated slip-outs, and more unstable tissues contain greater amounts of slipped-DNA compared to more stable tissues. This detection implies that slipped-DNA structures are not merely transient intermediates in the mutation and expansion process as has long been assumed, but remain within the DNA at detectable levels. The data reported herein both furthers our understanding of trinucleotide repeat instability, and additionally confirms the decades-long hypothesis that slipped-DNAs are in fact forming in patient tissues in a tissue-specific manner.

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

CTG repeat is one of the most common triplet repeat sequences that have been found to form slipped-strand structures leading to self-expansion during DNA replication. The lengthening of these repeats causes the onset of neurodegenerative diseases such as myotonic dystrophy. Through designing a series of CTG repeat sequences with high hairpin populations, systematic analysis of imino and methyl proton spectra study has been carried out to investigate the length and structural roles of CTG repeats in affecting the propensity of hairpin formation. Direct NMR evidence has been obtained to support three types of hairpin structures in sequences containing one to ten CTG repeats. The differences in loop structures and extent of interactions observed in the hairpins account for the differences in hairpin formation propensity and explain how slippage occurs that lead to triplet repeat expansion. / DNA has been found to adopt unusual structures leading to different types of mutations, which can ultimately cause genetic diseases and cancers. In this thesis, investigations on (i) structural role of CTG repeats in trinucleotide repeat expansion, (ii) primer-template structures in strand slippage during low fidelity replication and (iii) sequence effect of nucleotide downstream of thymine templates on primer-template structures have been carried out using NMR spectroscopy. / In addition, NMR structural investigations have also been carried out to determine solution structures of primer-template models. NMR evidence confirms misalignment can occur in primer-templates upon misincorporation of dNTP opposite a template sequence, leading to bulge formation in the primer-template. Depending on the template sequence, further incorporation of dNTP can bring about either realignment or further stabilization of the primer-template structure. Consequently, either mismatch or deletion errors will occur, leading to base substitution or frameshift mutation. These results imply that DNA sequences do not only play a passive role to store genetic information in the replication process, they also play an active structural role in governing the types of mutation during low-fidelity DNA replication. / Some of the results in this thesis have been reported in the following peer-reviewed journals: (1) Chi, L. M. and Lam, S. L. (2005) Structural roles of CTG repeats in slippage expansion during DNA replication. Nucleic Acids Res, 33, 1604-1617. (2) Chi, L. M. and Lam, S. L. (2006) NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication. FEBS Lett. , 580, 6496-6500. (3) Chi, L. M. and Lam, S. L. (2007) NMR investigation of primer-template models: structural effect of sequence downstream of a thymine template on mutagenesis in DNA replication. Biochemistry, 46, 9292-9300. / Chi, Lai Man. / "August 2007." / Adviser: Lam Sik Lok. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0877. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 102-112). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Trinucleotide repeat (TNR) expansions are the cause of over 40 human neurodegenerative diseases, and are linked to DNA damage and base excision repair (BER). We explored the role of DNA damage and BER in modulating TNR instability through analysis of DNA structures, BER protein activities, and reconstitution of repair using human BER proteins and synthesized DNA containing various types of damage. We show that DNA damage and BER can modulate TNR expansions by promoting removal of a TNR hairpin through coordinated activities of BER proteins and cofactors. We found that during repair in a TNR hairpin, coordination between the 5’-flap endonuclease activity of flap endonuclease 1 (FEN1), 3’-5’ exonuclease activity of AP endonuclease 1 (APE1), and activity of DNA ligase I (LIG I) can resolve the double-flap structure produced during BER in the hairpin loop. The resolution of the double-flap structure resulted in hairpin removal and prevention or attenuation of TNR expansions and provides the first evidence that coordination among BER proteins can remove a TNR hairpin. We further explored the role of BER cofactors in modulating TNR instability and found that the repair cofactor proliferating cell nuclear antigen (PCNA) facilitates genomic stability by promoting removal of a TNR hairpin. Hairpin removal was accomplished by altering dynamic TNR structures to allow more efficient FEN1 cleavage and DNA polymerase β (pol β) synthesis and stimulating the activity of LIG I. This study provides the first evidence that a DNA repair cofactor plays an important role in modulating TNR instability. Finally, we explored the effects of sugar modifications in abasic sites on activities of BER proteins and BER efficiency during repair in a TNR tract. We found that an oxidized sugar inhibits the activities of BER enzymes, interrupting their coordination and preventing efficient repair. Inefficient repair results in accumulation of repair intermediates with DNA breaks, contributing to genomic instability. Our results indicate that DNA base lesions and BER play a crucial role in modulating TNR instability. The research presented herein provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20788号 / 医博第4288号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 高橋 淳, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les répétitions de trinucléotides sont des séquences répétées en tandem pouvant subir, chez l'homme, de larges expansions à l'origine de nombreuses maladies génétiques. La dystrophie myotonique de type 1 (DM1) est due à l'expansion d'une répétition CTG en 3'UTR du gène DMPK. Les mécanismes d'instabilités des répétitions, peu connus, reposeraient sur leur capacité à former des structures secondaires constituant un obstacle aux mécanismes impliquant une synthèse d'ADN. Nous avons montré qu'une TALEN induisant une cassure double brin dans les répétitions CTG à l'origine de la DM1 insérées chez la levure Saccharomyces cerevisiae permettait de manière efficace et spécifique d'aboutir après réparation à leur contraction. Le mécanisme de réparation est dépendant uniquement de deux gènes, RAD50 et RAD52, suggérant la formation de structures aux extrémités de la DSB devant être retirées pour initier la réparation, suivis d'une réaction de SSA entre les répétitions aboutissant à leur contraction. L'efficacité et spécificité d'un système CRISPR-Cas9 à contracter ces répétitions chez la levure ont été comparées à la TALEN. L'induction de CRISPR-Cas9 n'aboutit pas à la contraction des répétitions mais à des réarrangements chromosomiques suggérant un manque de spécificité et un mécanisme de réparation différent de celui de la TALEN. Enfin, nous avons étudié si ces nucléases peuvent contracter ces répétitions CTG à des tailles non pathologiques dans des cellules de mammifères. L'induction de la TALEN dans des cellules de souris transgéniques DM1, puis dans des fibroblastes humains de patients DM1 montre des résultats préliminaires encourageant de contraction des répétitions. / Trinucleotides repeats are a specific class of microsatellites whose large expansions are responsible for many human neurological disorders. Myotonic dystrophy type 1 (DM1) is due to an expansion of CTG repeats in the 3’UTR of DMPK gene, which can reach thousands of repeats. Molecular mechanisms leading to these large expansions are poorly understood but in vitro studies have shown the capacity of these repeats to form secondary structures, which probably interfere with mechanisms involving DNA synthesis. We shown that a TALEN used to induce double-strand break (DSB) in DM1 CTG repeats integrated in the yeast Saccharomyces cerevisiae is specific and leads to highly efficient repeat contractions after repair. Mechanism involved in TALEN-induced DSB only depends of RAD50 and RAD52 genes, suggesting the formation of secondary structures at DSB ends that need to be removed for repair initiation, followed by an intramolecular recombinaison repair such as SSA between repeats leading to their contraction. We compared the efficiency and specificity of a CRISPR-Cas9 and the TALEN to contract CTG repeats in yeast. Surprisingly, CRISPR-Cas9 induction do not lead to repeat contraction but to chromosomal rearrangement, suggesting a lack of specificity and a different repair mechanism than with the TALEN. At last, we studied whether these nucleases could contract CTG repeats to a non-pathological length in mammalian cells. Finally, TALEN induction in DM1 transgenic mice cells, and in DM1 human fibroblasts show promising repeat contractions.

This dissertation examines the history of the creation and development of the concept of anticipation, a pattern of heredity found in several diseases (e.g. Huntington’s disease and myotonic dystrophy), in which an illness manifests itself earlier and often more severely in successive generations. It reconstructs major arguments in twentieth-century debates about anticipation and analyzes the relations between different research communities and schools of thought. Developments in cutting-edge medicine, biology, and genetics are analyzed; many of these developments were centered in Britain, but saw significant contributions by people working in France, Germany, Switzerland, the Netherlands and North America. Chapter one traces precursor notions in psychiatric and hereditarian thought from 1840 to the coining of the term ‘anticipation’ by the ophthalmologist Edward Nettleship in 1905. Key roles in the following chapters are played by several figures. Prior to World War II, these include: the neuropathologist F.W. Mott, whose advocacy during 1911- 1927 led to anticipation being called “Mott’s law”; the biometrician and eugenicist Karl Pearson, who opposed Mott on methodological and political grounds; and two politically and theoretically opposed Germans – Ernst Rüdin, a leading psychiatrist and eugenicist who came to reject anticipation, and Richard Goldschmidt, a geneticist who offered a peculiar Mendelian explanation. The British psychiatrist and human geneticist, Lionel Penrose, makes a first interwar appearance, but becomes crucial to the story after World War II due to his systematic dismissal of anticipation, which discredited the notion on orthodox Mendelian grounds. The final chapters highlight the contributions of Dutch neurologist Christiaan Höweler, whose 1980s work demonstrated a major hole in Penrose’s reasoning, and British geneticist Peter Harper, whose research helped demonstrate that expanding trinucleotide repeats accounted for the transgenerational worsening without contradicting Mendel and resurrected anticipation as scientifically legitimate. Reception of the concept of anticipation is traced across the century through the examination of textbooks used in different fields. This dissertation argues against established positions regarding the history of the concept, including claims that anticipation’s association with eugenics adequately explains the rejection of the notion after 1945. Rejected, in fact, by many eugenicists from 1912, anticipation was used by physicians until the 1960s.

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.