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.

Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.

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No. of bitstreams: 2 Tese - Marc Alexandre Duarte Gigonzac - 2016.pdf: 12763622 bytes, checksum: 3479eadda35402525c2387337a3a0d69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-02-02 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / X-Fragile Syndrome (FXS) is the leading cause of inherited intellectual disability in the world and the second of genetic etiology, with an estimated prevalence of 1/4000 men and 1/8000 women. The most common molecular mechanism in SXF is due to changes in the expression of the FMR1 gene, located in Xq27.3, due to CGG trinucleotide expansions in the promoter region and subsequent methylation of the gene. In spite of presenting consistent clinical findings, they are not exclusive, and the existence of carriers of alteration in the FMR1 gene without apparent clinical manifestations makes it impossible to diagnose SXF based only on the evaluation. In the present study, a methodological proposal for the molecular diagnosis of X-Fragile Syndrome was developed from the methylation-specific triple amplification of the promoter region of the FMR1 gene combined with capillary electrophoresis. Thirty-four patients with clinical indication of SXF were referred to a laboratory of the public health network. After extraction and quantification of the DNA, the samples were amplified in an optimized protocol and the products submitted to 36cm capillary electrophoresis to verify the amount of CGG repeats and the degree of DNA methylation of each sample. Pre-mutation (3%) and six complete mutations (18%) were detected, all of which revealed a high degree of methylation. Considering the clinical signs commonly presented, the patients were also analyzed for the occurrence of Autism Spectrum Disorder (ASD), which shadowing and overlapping the SXF, verifying that 100% of the individuals with complete mutation presented the phenotype. Thus, it was possible to observe small behavioral differences in the patients analyzed, indicating a lighter clinical picture regarding aspects of social interaction and stereotypies. Thus, the new methodological proposal allows to effectively determine the CGG trinucleotide expansions in FMR1 allowing an assertive diagnosis of SXF for the families of patients attended in the public health network in Goiás. / A Síndrome do X-Frágil (SXF) é a principal causa de deficiência intelectual herdável no mundo e a segunda de etiologia genética, com uma prevalência estimada de 1/4000 homens e 1/8000 mulheres. O mecanismo molecular mais comum na SXF é decorrente de alterações na expressão do gene FMR1, localizado em Xq27.3, devido a expansões trinucleotídicas CGG na região promotora e subsequente metilação do gene. Apesar de apresentar achados clínicos consistentes, os mesmos não são exclusivos, e a existência de portadores de alteração no gene FMR1 sem manifestações clínicas aparentes impossibilitam o diagnóstico da SXF baseado apenas no exame físico. No presente estudo foi desenvolvido uma proposta metodológica para o diagnóstico molecular da Síndrome do X-Frágil a partir da amplificação tripla metilação-específica da região promotora do gene FMR1 combinada a eletroforese em capilar. Foram utilizados 34 pacientes com indicação clínica de SXF encaminhados para um laboratório da rede pública de saúde. Após extração e quantificação do DNA, as amostras foram amplificadas em protocolo otimizado e os produtos submetidos a eletroforese em capilar de 36cm para verificar a quantidade de repetições CGG e o grau de metilação do DNA de cada amostra. Foram detectadas uma pré-mutação (3%) e seis mutações completas (18%), sendo que todas estas últimas revelaram um alto grau de metilação. Considerando os sinais clínicos comumente apresentados, os pacientes foram também analisados para a ocorrência do Transtorno do Espectro do Autismo (TEA), que sombreia e se sobrepõe à SXF, verificando que 100% dos indivíduos com mutação completa apresentaram o fenótipo. Foi possível observar pequenas diferenças comportamentais nos pacientes analisados, indicando um quadro clínico mais leve quanto aos aspectos da interação social e das estereotipias. Sendo assim, a nova proposta metodológica permite determinar de forma eficaz as expansões trinucleotídicas CGG no FMR1 permitindo um diagnóstico assertivo da SXF para as famílias de pacientes atendidos na rede pública de saúde em Goiás.

A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

The polyglutamine diseases are a clinically heterogeneous group of inherited neurodegenerative disorders caused by expansion of polyglutamine-encoding (CAG)n trinucleotide repeats within the disease genes. It is increasingly clear that the amino acid sequences flanking the polyglutamine expansion in each disease protein, i.e. the specific protein context, contribute to selective neuronal toxicity by influencing the behavior of the disease protein within selectively vulnerable neuronal populations. In the studies described here, I explore the role that protein context plays in the polyglutamine disease, Spinocerebellar ataxia type 3 (SCA3). Toward this end, I utilize biochemical, cell-based, and animal models to gain a broader understanding of the SCA3 disease protein, ataxin-3, and generate tools for further exploration of the molecular properties of ataxin-3 that modulate its toxicity during disease. In Chapter 1, I provide an overview of the recognized polyglutamine diseases, emphasizing the elements of protein context that are distinct among the polyglutamine disease proteins and may contribute to the neuropathological and clinical heterogeneity within this family of diseases. Alternative splicing of the polyglutamine disease gene products adds an additional level of complexity to the tissue-specific protein context of expanded polyglutamine, yet this phenomenon has been underinvestigated. In Chapter 2, I examine the significance of ataxin-3 splice variation. Several minor 5' variants and both known 3' splice variants of ataxin-3, a deubiquitinating enzyme, are expressed at the mRNA level in brain. At the protein level, however, the C-terminal splice isoform with three ubiquitin interacting motifs (3UIM ataxin-3) is the predominant isoform in brain, independent of age or (CAG)n expansion. Although both C-terminal ataxin-3 splice isoforms display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and is more rapidly degraded by the proteasome. These observations demonstrate how alternative splicing of sequences distinct from the polyglutamine-encoding (CAG)n repeat can alter disease-related components of protein context. Knock-in models of polyglutamine diseases utilize pathogenic (CAG)n expansions within the endogenous genomic, transcript, and protein context to recreate key features of individual polyglutamine diseases. In chapter 3, I describe the creation of the first knock-in mouse model of SCA3. Hemizygous knock-in mice transmit the knock-in allele in Mendelian ratios and broadly express both the expanded Atxn3(Q3KQ82) protein and the wildtype murine Atxn3(Q6) protein. In this chapter, I also compare the gene targeting efficiencies and rates of chromosomal instability of a novel C57BL/6J ES cell line (UMB6JD7) and two well established ES cell lines (W4 and Bruce4.G9). Of these, Bruce4.G9 ES cells proved superior based on lower rates of aneuploidy and the production of germline transmitting chimeras. Finally, in Chapter 4 I discuss questions and concepts raised during the course of these studies, and suggest avenues of future research aimed at broadening our understanding of ataxin-3 physiology and of protein context-dependent elements in polyglutamine disease pathogenesis.

The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

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

The integrity of an organism's genome depends on the fidelity of DNA replication and the efficiency of DNA repair. The DNA mismatch repair (MMR) system, which is highly conserved from prokaryotes to eukaryotes, plays an important role in maintaining genome stability by correcting base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication and other DNA transactions. Mismatch recognition is a critical step in MMR. Two mismatch recognition proteins, MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer), have been identified in eukaryotic cells. MutSα and MutSβ have partially overlapping functions, with MutSα recognizing primarily base-base mismatches and 1-2 nt ID mispairs and MutSβ recognizing 2-16-nt ID heteroduplexes. The goal of this dissertation research was to understand the mechanism underlying differential mismatch recognition by human MutSα and MutSβ and to characterize the unique functions of human MutSα and MutSβ in MMR. In this study, recombinant human MutSα and MutSβ were purified. Binding of the proteins to a T-G mispair and a 2-nt ID mispair was analyzed by gel-mobility assay; ATP/ADP binding was characterized using a UV cross-linking assay; ATPase activity was measured using an ATPase assay; MutSα amd MutSβ’s mismatch repair activity was evaluated using a reconstituted in vitro MMR assay. Our studies revealed that the preferential processing of base-base and ID heteroduplexes by MutSα and MutSβ respectively, is determined by the significant differences in the ATPase and ADP binding activities of MutSα and MutSβ, and the high ratio of MutSα:MutSβ in human cells. Our studies also demonstrated that MutSβ interacts similarly with a (CAG)n hairpin and a mismatch, and that excess MutSβ does not inhibit (CAG)n hairpin repair in vitro. These studies provide insight into the determinants of the differential DNA repair specificity of MutSα and MutSβ, the mechanism of mismatch repair initiation, and the mechanism of (CAG)n hairpin processing and repair, which plays a role in the etiology and progression of several human neurological diseases.

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

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