Les oxydations sur les bases nucléiques constituent l'une des sources principale d'apparition de lésions sur l'ADN, qui peuvent être mutagènes ou létales pour les cellules en l'absence de réparation de l'ADN. La Formamidopyrimidine-ADN glycosylase (Fpg), une enzyme procaryote du système de réparation de l'ADN par excision de base (BER), initie la réparation d'un large panel de lésions de ce type via ses activités ADN glycosylase (excision de la base oxydée) et AP lyase (clivage du site abasique par β,δ-élimination). Nous avons réalisé des études fonctionnelles par des techniques biochimiques et structurales par cristallographie des rayons X afin de préciser la spécificité de substrat et le mécanisme catalytique de Fpg. Ainsi, nous avons pu mettre en évidence des déterminants structuraux permettant à cette enzyme d'accommoder des lésions de tailles très différentes dans son site actif, en l'occurrence des résidus 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) substitués ou non en N7 par des adduits encombrants. D'autre part, nous avons caractérisé structuralement et fonctionnellement la reconnaissance et l'excision par Fpg d'une lésion pyrimidique, la 5-hydroxy-5-méthyle-hydantoïne (Hyd). Ainsi, nous avons montré que cette lésion appariée à une cytosine était un bon substrat pour l'enzyme, et nous avons précisé structuralement le mode de reconnaissance de l'Hyd par Fpg. D'autre part, nous avons mis en évidence un comportement inattendu de l'enzyme sur ce substrat. En l'occurrence, nous avons montré biochimiquement et structuralement qu'un pontage covalent se formait en quantités non négligeables entre Fpg et l'Hyd dans des conditions physiologiques. Mots clés : Réparation de l'ADN; Réparation par excision de base; Formamidopyrimidine-ADN glycosylase; 2,6- diamino-4-hydroxy-5-formamidopyrimidine; 7,8-dihydro-8-oxo-guanine; 5-hydroxy-5-méthyle-hydantoïne.

<p>The serendipitous discovery of the potent cytotoxic properties of cisplatin brought about a revolution in the treatment of certain types of cancer, but almost fifty years later, there still remain unknown areas in the chemistry of cisplatin. There are questions regarding which form of the drug reaches its DNA target, or why certain DNA sequences are more preferred than others for reaction with cisplatin. The work presented here aims to address some of these problems, using quantum chemical calculations to complement and interpret available experimental data.</p><p>Cisplatin's activation reactions are explored by Density Functional Theory (DFT) on two model systems, one solely using a self-consistent reaction field (SCRF) for modeling bulk water, and one including an additional partial solvation shell of water molecules. It is concluded that adding explicit solvation provides a better picture than using SCRF solvation alone. The energy surface supports the view that the active form of cisplatin is the monoaquated form.</p><p>The activation reactions of the cisplatin-derived drug, JM118, are investigated using DFT and SCRF calculations using three solvation model systems. The results show a slower rate of hydrolysis for the first reaction, and a faster rate for the second, suggesting diaquated JM118 as the main DNA binding form of the drug.</p><p>Diaquated cisplatin's first and second reaction with guanine and adenine are studied using DFT and SCRF solvation. Cisplatin's propensity toward guanine in the first substitution is explained by larger stabilisation energy for the initially formed complex and by favoured kinetics. For the second substitution, higher stability in complexation with guanine over adenine is ascribed as the main factor favouring guanine over adenine substitution. This provides the first explanation for the predominance of 1,2-d(GpG) over 1,2-d(ApG) adducts, and the direction specificity of the 1,2-d(ApG) adducts.</p>

The serendipitous discovery of the potent cytotoxic properties of cisplatin brought about a revolution in the treatment of certain types of cancer, but almost fifty years later, there still remain unknown areas in the chemistry of cisplatin. There are questions regarding which form of the drug reaches its DNA target, or why certain DNA sequences are more preferred than others for reaction with cisplatin. The work presented here aims to address some of these problems, using quantum chemical calculations to complement and interpret available experimental data. Cisplatin's activation reactions are explored by Density Functional Theory (DFT) on two model systems, one solely using a self-consistent reaction field (SCRF) for modeling bulk water, and one including an additional partial solvation shell of water molecules. It is concluded that adding explicit solvation provides a better picture than using SCRF solvation alone. The energy surface supports the view that the active form of cisplatin is the monoaquated form. The activation reactions of the cisplatin-derived drug, JM118, are investigated using DFT and SCRF calculations using three solvation model systems. The results show a slower rate of hydrolysis for the first reaction, and a faster rate for the second, suggesting diaquated JM118 as the main DNA binding form of the drug. Diaquated cisplatin's first and second reaction with guanine and adenine are studied using DFT and SCRF solvation. Cisplatin's propensity toward guanine in the first substitution is explained by larger stabilisation energy for the initially formed complex and by favoured kinetics. For the second substitution, higher stability in complexation with guanine over adenine is ascribed as the main factor favouring guanine over adenine substitution. This provides the first explanation for the predominance of 1,2-d(GpG) over 1,2-d(ApG) adducts, and the direction specificity of the 1,2-d(ApG) adducts.

Cancer is the second leading cause of death in the United States. Cisplatin is one of the well-known anti-cancer agents used to treat testicular and ovarian cancers. It mainly binds to the DNA bases, which leads to cell death. The cytotoxic activity of the cisplatin analogs is due to the interaction of platinum with nucleotides like adenine at N7 or N1 position and guanine at N7 position. Guanine is the primary target for cisplatin analogs whereas adenine is the secondary target. Cisplatin analogs, [Pt(Me5dien)(D2O)]2+[Me5dien = N,N,N’,N’,N’’-pentamethyl diethylene triamine] and [Pt(dien)(D2O)]2+[dien=diethylene triamine] were synthesized and their effects on AMP and GMP were studied using NMR spectroscopy. The experiments were conducted to examine the effects of bulk on 5’-GMP and 5’-AMP. The results suggest that bulk slows down the reaction with AMP more than with that of GMP. The order of reactivity is Pt(dien)(GMP)> Pt(dien)(AMP) > Pt(Me5dien)(GMP) > Pt(Me5dien)(AMP). The reaction of the [Pt(Me5dien)(D2O)]2+ complex with AMP leads to multiple products, some of which appear to be due to coordination at N1 instead of N7.

Lfc is a guanine nucleotide exchange factor (GEF) for RhoA and is negatively regulated by its association with the microtubule array. Tctex-1, a light chain subunit of the dynein motor complex, was identified as an Lfc-interacting protein in a yeast two-hybrid screen. In mouse embryonic fibroblast (MEF) cells, over-expression of Tctex-1 represses Lfc-induced actin stress fiber and focal adhesion complex formation. Here, we present biochemical evidence obtained from a real-time, nuclear magnetic resonance (NMR)-based assay indicating that the microtubule exerts its inhibitory effect on Lfc through a mechanism that is dependent on the presence of Tctex-1. We also present NMR structure data showing that Lfc and the dynein intermediate chain (DIC) bind to different surfaces of Tctex-1. The biochemical and structural data together support a model in which Lfc is recruited to the microtubules through the dynein cargo adaptor function of Tctex-1, resulting in inhibition of Lfc function.

Lfc is a guanine nucleotide exchange factor (GEF) for RhoA and is negatively regulated by its association with the microtubule array. Tctex-1, a light chain subunit of the dynein motor complex, was identified as an Lfc-interacting protein in a yeast two-hybrid screen. In mouse embryonic fibroblast (MEF) cells, over-expression of Tctex-1 represses Lfc-induced actin stress fiber and focal adhesion complex formation. Here, we present biochemical evidence obtained from a real-time, nuclear magnetic resonance (NMR)-based assay indicating that the microtubule exerts its inhibitory effect on Lfc through a mechanism that is dependent on the presence of Tctex-1. We also present NMR structure data showing that Lfc and the dynein intermediate chain (DIC) bind to different surfaces of Tctex-1. The biochemical and structural data together support a model in which Lfc is recruited to the microtubules through the dynein cargo adaptor function of Tctex-1, resulting in inhibition of Lfc function.

5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogue, is genotoxic and teratogenic. The exposure of mouse embryos to BrdU at doses that cause malformations induces oxidative stress and an embryonic stress response characterized by an increase in c-Fos dependent AP-1 DNA binding. The goal of this thesis was to test the hypothesis that development is disturbed at sites where BrdU is incorporated into DNA, triggering oxidative stress and c-Fos induction. Gestation day 9 CD-1 mice were treated with BrdU and embryos were obtained for immunolocalization of BrdU, 8-oxoguanine, a biomarker for oxidative stress, and c-Fos. BrdU incorporation into DNA was dispersed throughout the embryo. In contrast, the staining for 8-oxoguanine and c-Fos were highest in the neuroepithelium. BrdU incorporation was not affected by the pre-administration of N-acetyl-cysteine (NAC), an anti-oxidant, although both 8-oxoguanine and c-Fos staining were decreased. Thus, the response of the embryo to insult is tissue specific.

Les oxydations sur les bases nucléiques constituent l'une des sources principale d'apparition de lésions sur l'ADN, qui peuvent être mutagènes ou létales pour les cellules en l'absence de réparation de l'ADN. La Formamidopyrimidine-ADN glycosylase (Fpg), une enzyme procaryote du système de réparation de l'ADN par excision de base (BER), initie la réparation d'un large panel de lésions de ce type via ses activités ADN glycosylase (excision de la base oxydée) et AP lyase (clivage du site abasique par ß,d-élimination). Nous avons réalisé des études fonctionnelles par des techniques biochimiques et structurales par cristallographie des rayons X afin de préciser la spécificité de substrat et le mécanisme catalytique de Fpg. Ainsi, nous avons pu mettre en évidence des déterminants structuraux permettant à cette enzyme d'accommoder des lésions de tailles très différentes dans son site actif, en l'occurrence des résidus 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) substitués ou non en N7 par des adduits encombrants. D'autre part, nous avons caractérisé structuralement et fonctionnellement la reconnaissance et l'excision par Fpg d'une lésion pyrimidique, la 5-hydroxy-5-méthyle-hydantoïne (Hyd). Ainsi, nous avons montré que cette lésion appariée à une cytosine était un bon substrat pour l'enzyme, et nous avons précisé structuralement le mode de reconnaissance de l'Hyd par Fpg. D'autre part, nous avons mis en évidence un comportement inattendu de l'enzyme sur ce substrat. En l'occurrence, nous avons montré biochimiquement et structuralement qu'un pontage covalent se formait en quantités non négligeables entre Fpg et l'Hyd dans des conditions physiologiques.

Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 144-178). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Watson-Crick paired B-form DNA is the genetic material in most of the biological systems. Integrity of DNA is of utmost importance for the normal functioning of any organism. Various environmental factors, chemicals and endogenous agents constantly challenge integrity of the genome resulting in mutagenesis. Over the past few decades multiple reports suggest that DNA can adopt alternative conformations other than the right handed double helix. Such structures occur within the context of B-DNA as sequence dependent structural variations and are facilitated by free energy derived from negative supercoiling, which may be generated during physiological processes like transcription, replication, etc. or binding of proteins. Multiple groups have shown that these structures render fragility to the genome owing to single-strandedness (presence of unpaired bases). This conformational polymorphism of the DNA is due to the presence of several repetitive elements across the genome. Some of the common non-B DNA structures include Z-DNA, H-DNA (triplex DNA), cruciform DNA, G-quadruplexes and RNA: DNA hybrid (R-loops). Over the past few decades G-quadruplex structures have gained tremendous importance owing to its role in physiology and pathology. Recently it has been shown that novel sequence motifs, called GNG or bulges can fold into G-quadruplexes, thus increasing the propensity of such structures genome-wide. Neurological diseases, psychiatric diseases and genomic disorders (due to deletions, translocations, duplications and inversions) are some of the consequences of non-B DNA structures in the human genome. Inadvertent genomic rearrangements in human can lead to different diseases including cancer. Immediate consequence of genomic rearrangement includes structural alteration of genome through joining of distant sequences. t(8;14) translocation is the hallmark of Burkitt’s lymphoma, which results in deregulation of c-MYC gene that may contribute to oncogenic transformation. In the present study, we delineate the causes of fragility within the c-MYC gene. In order to do this, breakpoints at the c-MYC locus from Burkitt’s lymphoma patient sequences reported in database were plotted and analysed. Interestingly, unlike many other translocations, breakpoints at c-MYC locus were widespread, except for a cluster of breakpoints downstream to promoter 2 (P2). Previous studies indicate that the translocation breakpoint clusters often correlate with formation of non-B DNA structures. The entire breakpoint cluster downstream of P2 was divided into Region 1, Region 2 and Region 3. Interestingly, in silico analysis of the breakpoint clusters revealed no evidence for predictive classic non-B DNA motifs in Region 2; whereas Region 1 harboured a G-quadruplex motif on the template strand and Region 3 had two short inverted repeats. Intriguingly, as the nontemplate strand of Region 2 was G skewed with a good number of AID binding motifs, we tested the MYC breakpoint Region 2 for its potential to form R-loop due to binding of nascent RNA to template DNA. Our results showed that MYC Region 2 can form RNA-DNA hybrid in a transcription dependent manner in physiological orientation. Observed structure was sensitive to RNase H. We showed Region 2 hindered action of Dpn I upon transcription confirming formation of R-loop structure. Owing to single strandedness, Region 2 R-loop was shown to be sensitive to P1 nuclease as opposed to the untranscribed control. The single strandedness of the Region 2 R-loop was characterized at a single molecule level through bisulfite modification assay. The assay corroborated formation of R-loop along with providing snapshots of various length R-loops formed upon Region 2 transcription. Besides, various biophysical and biochemical assays showed the complementary region (template strand) to be single-stranded in stretches, upon transcription. Length of RNA within the R-loop was within a range of 75 to 250 nt. To delineate the mechanism of R-loop formation we tested the sensitivity of R-loop formation to RNase A during and post transcription; and found that R-loop formation was abrogated in presence of RNase A during transcription suggesting that R-loop formation followed a “thread back model”. Intriguingly we observed that two short regions of the template strand exhibited high degree of single strandedness. To investigate the reason for such unusual single strandedness, oligonucleotides spanning the region was designed and subjected for CD and EMSA studies. EMSA showed robust intramolecular G-quadruplex structure formation in presence of KCl, whereas CD confirmed that both regions formed parallel G-quadruplexes. We also showed the precise involvement of guanines in structure formation through DMS protection assay. Further, the region of interest was cloned into appropriate vectors and primer extension assays were performed in presence of G-quadruplex stabilizing agents like TMPyP4 and KCl. Increasing concentration of these stabilizing agents enhanced the formation of G-quadruplexes in a double stranded context, which hindered polymerase progression. Since these G-quadruplex structures utilized sequences which are deviant to the consensus of G-quadruplex motifs, non-B DNA predicting tools were unable to score them. On closer analysis of the sequences we found that, these G-quadruplexes involve duplex hairpin and GNG motifs during structure formation. Besides, both the G-quadruplexes were highly thermostable and were able to fold back upon renaturation. Till recently, it has been believed that G-quadruplex structures are formed using a minimum of four, 3 guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In the present study, guanines along with GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the core elements of a G-quadruplex are not equally important in structure formation when flanked by GNG motifs. Importantly, the positioning and number of GNG/GNGNG can dictate the formation of G-quadruplexes. In addition to HOX11 fragile region, GNG motifs of HIF1-alpha can fold into intramolecular G-quartet. However, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX), it can fold into an intramolecular G-quadruplex. Interestingly, intra molecular GNG G-quadruplexes were able to fold back after complete denaturation of the oligonucleotides. Besides one of the intra molecular GNG G-quadruplexes was purified and confirmed for parallel conformation. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody BG4 to SHOX G-quadruplex through EMSA studies. Thus, the study provides novel insights into the role of GNG motifs in G-quadruplex structure formation, which may have both physiological and pathological implications. In conclusion, we show formation of transcription dependent R-loop and G-quadruplex structures at the c-MYC gene locus in a mutually exclusive manner. The data presented here, in conjunction with studies from other laboratories suggests that these structures could impart fragility within the c-MYC gene locus during t(8;14) translocation. Besides, we characterised unusual G-quadruplexes harbouring GNG motifs. We find that positioning and number of GNG can dictate the formation of G-quadruplexes and is context dependent.