Dans les trois domaines du vivant, que constituent les bactéries, les eucaryotes et les archées, une molécule a la capacité souveraine de gouverner la vie, la mère à l’origine de tous les mécanismes biologiques, l’ADN. S’il est évident de dire que le maintien de l’intégrité des génomes est essentiel à la vie, il existe deux systèmes qui le permettent, la réplication et la réparation de l’ADN. La fidélité de ces derniers est finement influencée par la disponibilité (ratio et balance) des précurseurs nucléotidiques désoxyribonucléotides (dNTPs) et ribonucléotides (rNTPs) au cours du cycle cellulaire. Même si la concentration intracellulaire en nucléotides est largement documentée chez les eucaryotes et les bactéries, ça n’est malheureusement pas le cas chez les archées. En ce qui concerne l’étude de la maintenance génomique, un groupe d’archées a intéressé les chercheurs de par leurs capacités à survivre dans des milieux dits extrêmes. Pyrococcus abyssi est l’une d’entre elles qui depuis de nombreuses années sert de modèle biologique pour répondre aux questions de la stabilité de l’ADN à haute température. Cette étude est centrée sur cette thématique et particulièrement sur les caractéristiques fonctionnelles des ADN polymérases: PolD, PolB et le complexe p41/p46. Initialement, le contenu en nucléotides a été évalué dans des cellules en phase exponentielle de croissance par la technique de chromatographie couplée à une double détection en spectrométrie de masse (zicHILIC-MS-MS). Les résultats montrent que le contenu en rNTPs est de 20 fois supérieur à celui en dNTPs. Pour cette raison, la discrimination sélective des dNTPs par les ADN polymérases est mise à l’épreuve. Même si, des mécanismes permettent d’exclure les rNMPs durant la synthèse de l’ADN, de récentes études ont montrées que des rNMPs étaient incorporés par des ADN pols. Ainsi, le ratio en nucléotides obtenu a été utilisé pour l’analyse de son effet sur la synthèse d’ADN par les ADN Pols et les extraits cellulaires de P. abyssi. Les résultats démontrent clairement que les rNMPs sont incorporés par l’ADN polymérase PolD. Puis, les conséquences de la présence des rNMPs dans l’ADN sur la réplication ont été étudiées et ont mis en évidence que les extraits cellulaires, tout comme les ADN Pols de P. abyssi étaient capables de « passer » un rNMP présent dans l’ADN. Pour finir, une étude de l’incorporation préférentielle de chaque dNMP et rNMP a été menée démontrant que la complémentarité des bases était respectée même lors de l’incorporation de rNMPs. Enfin, la caractérisation de la petite sous-unité, DP1, de PolD a permis de montrer sa capacité à retirer des rNMPs grâce à son activité de relecture, suggérant un premier rempart à la présence de rNMPs dans l’ADN. Pour conclure, ces résultats montrent que la présence de rNMPs dans l’ADN est un phénomène conservé dans les trois domaines du vivant. / In the three domains of life that include Bacteria, Eukarya and Archaea, one molecule has the sovereign ability to govern life, and not the least one, the mother of all biological mechanisms, DNA. Maintaining the integrity of genomes is obviously essential for life, and faithful DNA replication and repair are the guarantees. The fidelity of these two processes may vary depending on the availability and levels (balance and ratio) of deoxyribonucleotides (dNTPs) and ribonucleotides (rNTPs) during the cell-cycle. Even if intracellular concentration of nucleotides is largely documented in Eukarya and Bacteria, it remains limited in Archaea. From many years one group of Archaea is of great interest for studying genomic maintenance, because of its ability to survive in extremes environments. Pyrococcus abyssi is one of them that is used as biological model for deciphering the stability of DNA at elevated temperature in LM2E. The present work focuses on genomic integrity and particularly on the functional characterization of the three DNA polymerases: PolD, PolB and the p41/p46 complex. Initially, the nucleotide pool has been evaluated in exponentially growing cells using the highly sensitive method that combined chromatography and mass spectrometry (zicHILIC-MS-MS). The results show that rNTPs content is 20-fold higher than dNTPs. For that reason, fidelities of DNA polymerases are challenged to select the correct dNTP over the most abundant rNTP during DNA synthesis. Despite the fact that some mechanisms allow the exclusion of rNTPs from entry to the Pol active site, recent findings indicate that ribonucleotides are incorporated by different DNA Pols with surprisingly high frequency. In this work, the obtained intracellular balance and ratio of rNTPs and dNTP have been used to analyze their effect on DNA synthesis by P. abyssi DNA Pols and cell-free extracts. Our results clearly demonstrate that rNTP incorporation is detectable with distinct efficiencies among DNA pols. Secondly, the consequences of the presence of rNMPs in a DNA template on DNA polymerisation has been examined and highlights that cell-free extracts are able to bypass a single rNMP as well as replicative DNA polymerases. To strengthen that study, single nucleotide incorporation opposite rNMP or dNMP has been carried out and the results demonstrate that replicative Pyrococcus abyssi DNA Pols can basepair the complementary rNTPs opposite dNMPs, and vice-versa, the complementary dNTPs opposite rNMPs.Furthermore, the preliminary results obtained about the nucleolysis activities of the PolD small subunit, DP1, show that the DNA polymerase D is able to remove rNMPs from a DNA strand, suggesting a first level of protection against ribonucleotide contamination of DNA. Definitely, these data indicate that the presence of transient embedded rNMPs in genomic DNA represents a universally conserved phenomenon across Archaea, Bacteria and Eukarya.

DNA polymerases are a family of molecular machines involved in high-fidelity DNA replication and repair, of which DNA polymerase I (Pol) is one the best-characterized members. Pol is a strand-displacing polymerase responsible for Okazaki fragment synthesis and base-excision repair in bacteria; it consists of three protein domains, which harbour its 5’-3' polymerase, 3’-5’ exonuclease and 5’ endonuclease activities. In the first part of the thesis, we use a combination of single-molecule Förster resonance energy transfer (smFRET) and rigid-body docking to probe the structure of Pol bound to its gapped-DNA substrate. We show that the DNA substrate is highly bent in the complex, and that the downstream portion of the DNA is partly unwound. Using all-atom molecular dynamics (MD) simulations, we identify residues in the polymerase important for strand displacement and for downstream DNA binding. Moreover, we use coarse-grained simulations to investigate the dynamics of the gapped-DNA substrate alone, allowing us to propose a model for specific recognition and binding of gapped DNA by Pol. In the second part of the thesis, we focus on the catalytically important conformational change in Pol that involves the closing of the ‘fingers’ subdomain of the protein around an incoming nucleotide. We make use of the energy decomposition method (EDM) to predict the stability-determining residues for the closed and open conformations of Pol, and test their relevance by site-directed mutagenesis. We apply the unnatural amino acid approach and a single-molecule FRET assay of Pol fingers-closing, to show that substitutions in the stability-determining residues significantly affect the conformational equilibrium of Pol. In the final part of the thesis, we attempt to study Pol in its native environment of the living cell. We make use of the recently developed method of internalization by electroporation, and optimize it for organically labelled proteins. We demonstrate the internalization and single-molecule tracking of Pol, and provide preliminary data of intra-molecular FRET in Pol, both at the single-cell and single-molecule levels. Finally, by measuring smFRET within an internalized gapped-DNA construct, we observe DNA binding and bending by endogenous Pol, confirming the physiological relevance of our in vitro Pol-DNA structure.

No description available.

Submitted by Maria Eneide de Souza Araujo (mearaujo@ipen.br) on 2017-03-10T17:07:28Z No. of bitstreams: 0 / Made available in DSpace on 2017-03-10T17:07:28Z (GMT). No. of bitstreams: 0 / Este estudo teve como objetivo avaliar a adaptação marginal em restaurações classe II em dentina e esmalte dental utilizando um sistema adesivo universal de acordo com três técnicas diferentes de aplicação: Autocondicionante, condicionamento seletivo e condicionamento total, através do sistema de tomografia por coerência óptica (OCT), antes e após ao envelhecimento por termociclagem (TC). Preparos classe II foram confeccionados nas paredes mesial e distal, em 30 molares hígidos humanos, com término da cavidade em esmalte e em dentina. Todos os espécimenes foram restaurados (adesivo Single Bond Universal e resina composta Filtek Z350 XT) nas três técnicas de aplicação. Os dentes foram distribuídos aleatoriamente em três grupos (n=10), sendo um para cada técnica de aplicação adesiva: Grupo I: autocondicionante, Grupo II: condicionamento seletivo e Grupo III: condicionamento total; em seguida os grupos foram avaliados antes e depois a termociclagem pelo sistema de tomografia por coerência ótica. De posse dos corpos de prova devidamente restaurados, partimos para os procedimentos de termociclagem (1000 ciclos de 1 minuto com intervalo de 30 segundos) e a avaliação pelo sistema de Tomografia por Coerência Óptica. Em seguida, as amostras foram examinadas por meio de imagens geradas pelo OCT e os dados foram submetidos ao teste estatístico não-paramétricos de Kruskal-Wallis e Dunn (p<0.05). Foi observada diferença estatisticamente significante da adaptação marginal entre os grupos com término em esmalte (p= 0.0073); para os grupos com término em dentina, não foi observada diferença estatisticamente significante na adaptação marginal (p=0.2063). Conclui-se que o OCT foi, então, capaz de diagnosticar a microinfiltração marginal nas restaurações em cavidades classe II. Existe deterioração das margens e alteração de padrão de infiltração marginal com as diferentes técnicas adesivas dos preparos classe II restauradas com resina em esmalte. Atribui-se à termociclagem a falha de vedamento marginal observada nos espécimes após em esmalte. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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.

To maintain the genomic integrity, cell has evolved various DNA repair pathways. Base Excision Repair pathway (BER) is one such DNA repair pathway which is dedicated to protect DNA from small lesions such as oxidation, alkylation, deamination and loss of bases. Uracil is a promutagenic base which appears in the genome as a result of misincorporation of dUTP or due to oxidative deamination of cytosine. Uracil-DNA glycosylases (UDGs) are DNA repair enzymes that initiate multistep base excision repair (BER) pathway to excise uracil from DNA. Excision of uracil generates an abasic site (APDNA). AP-sites are cytotoxic and mutagenic to the cell. AP endonucleases act downstream to UDG in this pathway and generate substrates for DNA polymerase to fill in the correct bases. The cytotoxicity of AP-sites raises the question whether uracil excision activity is coupled to AP endonuclease activity. Also, there is transient formation of single stranded DNA (ssDNA) during DNA metabolic processes such as replication, repair and recombination. ssDNA is more prone to various nucleases and DNA damaging agents. All the living organisms encode single stranded DNA binding protein (SSB) that binds to ssDNA and protects it from various damages. In addition, SSB plays a vital role during DNA replication, repair and recombination. Studies on SSBs from prototype Escherichia coli and an important human pathogen, Mycobacterium tuberculosis have shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. My PhD thesis consists of four Chapters. Chapter 1 summarizes the relevant literature review on DNA damage and repair with an emphasis on uracil DNA glycosylase and its interacting protein, SSB. Chapters 2 and 3 describe my studies on the mechanism of uracil excision repair in E. coli. Chapter 4 describes my findings on the structure-function relationship of single stranded DNA binding proteins from E. coli and M. tuberculosis. Specific details of my research are summarized as follows: (1) Analysis of the impact of allelic exchange of ung with a mutant gene encoding Uracil DNA Glycosylase attenuated in AP-DNA binding in the maintenance of genomic integrity in Escherichia coli. There are five families of UDGs. Of these, Ung proteins (family 1 UDGs) represent highly efficient and evolutionary conserved enzymes. Structural and biochemical analysis of Ung proteins has identified two conserved motif, motif A (62GQDPY66) and motif B (187HPSPLS192) in E. coli that are important for the catalysis by Ung enzyme. Y66 of motif A is in van der Waals contact with the C5 position of the uracil and prevents entry of other bases. Earlier study from the laboratory showed that the Y66W and Y66H mutants of Ung were compromised by ~7 and ~170 fold, respectively in their uracil excision activities. However, unlike the wild-type and Y66H proteins, Y66W was not inhibited by its product (uracil or AP-DNA). In this study, by fluorescence anisotropy measurements I have shown that compared with the wild-type protein, the Y66W mutant is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in E. coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed ~5, ~3.0 and ~2.0 fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region (RRDR) of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. These findings have been discussed in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair. It is proposed that an error prone replication against AP-sites (as a result of uracil excision activities on A:U pair) may result in A to G mutations. 2. Mechanism of appearance of A to G mutations in ungY66W:amp strain of Escherichia coli. In this part of my study, I have investigated the role of error prone DNA polymerases in the mutational specificity of ungY66W:amp strain. It was observed from various studies in E. coli that, DNA polymerase IV (Pol IV) and DNA polymerase V (Pol V) are involved in error-prone replication on damaged or AP-site containing DNA. E. coli strains containing deletion of either dinB (encoding DNA Pol IV) or umuDC (encoding DNA Pol V) were generated and used to study mutation frequency and mutation spectrum. Deletion of DNA Pol V resulted in a decrease in A to G mutations in ungY66W:amp E. coli strain, suggesting that increase in A to G mutations were a consequence of error prone incorporation by DNA Pol V. 3. Structure and Function studies on Single Stranded DNA Binding Proteins from Escherichia coli and Mycobacterium tuberculosis. SSB from M. tuberculosis (MtuSSB) has similar domain organization as the EcoSSB. Moreover, the biochemical properties such as oligomerization, DNA binding affinity and minimum binding site size requirements were shown to be similar to EcoSSB. However, structural studies suggested that quaternary structures of these two SSBs are variable. In this study I have used X-ray crystal structure information of these two SSBs to generate various chimeras after swapping at various regions of SSBs. Chimeras mβ1, mβ1’β2, mβ1-β5, mβ1-β6, and mβ4-β5 SSBs were generated by substituting β1 (residues 611), β1’β2 (residues 21-45), β1-β5 (residues 1 to 111), β1-β6 including a downstream sequence (residues 1 to 130), and β4-β5 (residues 74-111) regions of EcoSSB with the corresponding sequences of MtuSSB, respectively. Additionally, mβ1’β2ESWR SSB was generated by mutating the MtuSSB specific ‘PRIY’ sequence in the β2 strand of mβ1’β2 SSB to EcoSSB specific ‘ESWR’ sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) efficiently bound DNA in modes corresponding to limited and unlimited modes of binding. The mβ1 SSB was also hypersensitive to chymotrypsin treatment. The mβ1-β6, MtuSSB, mβ1’β2 and mβ1-β5 constructs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1’β2ESWR SSB complemented E. coli as well as EcoSSB. Interestingly, the inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Indiana University-Purdue University Indianapolis (IUPUI) / Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.

Indiana University-Purdue University Indianapolis (IUPUI) / Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.

Indiana University-Purdue University Indianapolis (IUPUI) / Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.