Lors de la vie, des mécanismes de réparation de l’ADN sont mis en oeuvre lors d’agressions, pour protéger le génome. La réparation par excision de nucléotides (NER) est l’un de ces mécanismes. Des mutations des facteurs NER sont à l’origine de 3 maladies génétiques humaines: Xeroderma pigmentosum (XP), la trichothiodystrophie (TTD) et le syndrome de Cockayne (CS). Certains de leurs signes cliniques ne sont pas expliqués par un défaut de réparation de l’ADN. Des études suggèrent que ces facteurs interviennent dans d’autres processus, notamment lors de l’expression des gènes. Durant ma thèse, je me suis intéressé aux rôles des facteurs NER dans la transcription. En effet, j’ai montré que ces facteurs, dit de réparation, étaient recrutés avec la machinerie transcriptionnelle au niveau du promoteur et du terminateur de gènes activés. Ils influencent l’environnement chromatinien des gènes activés (boucles de chromatine et modifications post-­‐ traductionnelles des histones). Ma thèse apporte une meilleure compréhension du processus de transcription des gènes activés, permettant de mieux comprendre certaines anomalies associées aux yndromes XP, CS et TTD. / Throughout life, the mechanisms of DNA repair are implemented in attacks to protect the integrity of our DNA. The nucleotide excision repair (NER) is one of these mechanisms. Mutations targeting genes of NER factors (XPA-­‐G, TTD-­‐A, CSA and CSB) are responsible for three human genetic diseases : Xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Some of them clinical features cannot be explained by a defect in DNA repair only. Previous studies suggest that these factors could be involved in other functions, including gene expression. In my thesis, I am interested in the roles of NER factors during the transcription process. Indeed, we have shown that these “repair” factors, were recruited with the transcription al machinery at the promoter and terminator of activated genes during transcription. They influence the chromatin environment of activated genes (chromatin loops and post-­‐translational modifications of histones).My thesis provides a better understanding of the transcription process of activated genes and allows a better understanding of some syndromes associated with XP, CS and TTD.

Maintaining the chromosome continuity and complete genetic information in human cells is crucial for cell survival and the whole organism. It prevents life-threatening pathologies and preserves genetic continuity. However, cellular DNA is exposed to both endogenous and exogenous stress damaging its content and integrity. This stress activates mechanisms involving detection and repair of these damaged sites (DDR). One of the most serious types of DNA damage double-stranded breaks (DSB) occuring when both strands are severed. DSBs trigger wave of PTMs that regulate protein interactions, nuclear localization and catalytic activity of hundreds of proteins. Such modifications include acetylation, methylation, SUMOylation, ubiquitinylation and especially phosphorylation. The most important kinases involved in DDR kinases are ATM, ATR and DNA-PK. These kinases are activated immediately after the detection of the damaged area. DAXX (Death-associated protein 6) is an adapter and predominantly nuclear protein, which is involved in chromatin remodeling, gene expression modulation, antiviral response and depositing histone H3.3 variants into chromatin or telomeres. Daxx is essential for murine embryogenesis, since the homozygous deletion is lethal in E9.5-10. In 2006 a study mapping the substrates of kinases...

Gram-positive pathogenic bacteria utilize cell-surface anchored proteins to bind and transport heme into the cell. These bacteria acquire iron from host proteins containing heme e.g., hemoglobin. Proteins like HmuT from Corynebacterium diphtheriae bind and help transport heme into the cell. Residues His136 and Tyr235 are utilized as the axial ligands, with a conserved Arg237 residue acting as the hydrogen bonding partner to the axial Tyr235. Similarly, Streptococcus pyogenes utilizes the cell anchored protein Shr to transfer heme into the cell. Shr-NEAT2 is hexacoordinated by two axial methionines and is prone to autoreduction where lysines are the most likely source of electrons. Lastly, PefR of Group A Streptococcus is a DNA transcription factor which regulates protein expression. Preliminary studies indicate a cysteine may coordinate the heme. A combination of UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies shows these proteins play a crucial role heme transport and regulation.

The anti-tumor activity of platinum based drugs is mediated by their ability to attack DNA. Platinum complexes can alter the structure of DNA by modifying the bases, mainly guanines. The biological consequnces of such interactions are compromising replication and transcription. RNA polymerase complex can stall at a damaged site in DNA and mark the lesion for repair by proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Main goal of this thesis was to study RNA polymerases tolerance of DNA damage by novel, biologically active platinum (II) complexes involving derivatives of aromatic cytokinines as the ligands; cis-[Pt(2-chloro-6-(4-methoxybenzylamino)-9-isopropylpurin)2Cl2](PR-001), cis-[Pt(2-chloro-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-002 )and cis-[Pt(2-(3-hydroxypropylamino)-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-005). DNA templates (constructs) that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II and bacteriophage T7 RNA polymerase were prepared. The method is making use of polymerase chain reaction (PCR) and biotin-streptavidin interactions and paramagnetic particles to purify the final product. Synthetic oligomers duplexes (75-mer, 56-mer and 15-mer) are ligated to 5´-biotin pCI-neo-G-lessT7 PCR fragment, the 15-mer is either unmodified or modified with a site-specific lesion of PR-005 and cisplatin. We also studied the inhibition of RNA polymerases activity on globally modified plasmid pCI-neo and pUC 19 by novel platinum complexes and cisplatin. We found that bifunctional adducts of complex PR-005 contrary to adducts of PR-001 and PR-002 effectively decrease amount of full lenght transcripts produced by both, human and bacterial RNA polymerases. This result can be explained by a sterical block, induced to DNA by intrastrand cross-link of PR-005 with bulky aromatic ligands.

Packaging of genomic DNA by proteins and super coiling into chromatin and chromatin-like structures (in bacteria) influences nearly all nuclear process such as replication, transcription, repair, and recombination. A ubiquitous class of enzymes termed “DNA topoisomerases” pay key roles during these process. The reactions catalyzed by the members of the DNA topoisomerases family share a common chemistry, which involves phosphodiester bond breakage and re-joining, to bring about a change in the linking number of DNA. Nevertheless, the underlying mechanisms used by these enzymes differ significantly from another. Consequently, DNA topoisomerases are divided into type I and type II enzymes. The mechanism(s) by which DNA topoisomerases perform their functions, and act as targets for anti-bacterial and anti-neoplastic drugs, has attracted considerable interest. Based on these and other finding, I have chosen DNA gyrase from mycobacteria as the subject of my Ph.D. theses investigation. The prokaryotic enzyme, DNA gyrase, is unique amongst all topoisomerases being the only enzyme capable of introducing negative super coils in to duplex DNA. Since no equivalent enzymatic activity has been reported in humans, this essential enzyme has been exploited as a during target against many microbial infections including tuberculosis.DNA gyrase is a tetrameric protein, comprised of two pairs of subunits, encoded by gyrA and gyrB. Inhibitors of DNA gyrase know till date target either of the two subunits and are categorized broadly in to two class, viz. coumarins and quinolones. With the emergence of multiple-drug resistant strains of pathogenic bacteria such as Mycobacterium tuberculosis, which is a leading cause of death world-wide, there is a need to develops new lead molecules with novel mechanisms of inhibition. Towards this end, a new approach to inhibit the mycobacterial DNA gyrase using single-chain antibody has been explore in the present study. In addition to this, the differences in the catalytic properties of the subunits and assembly of the Mycobacterium smegmatis enzyme vis-à-vis Escherichia coli DNA gyrase have been examined. Further, the in vivo relationship of DNA gyrase with the transcription machinery of the cell has also been investigated, with an emphasis on the biology of mycobacteria.

This PhD thesis is focused on the synthesis of DNA modified with photocleavable 2- nitrobenzyl protecting groups in major groove and its applications in the regulation of gene expression in the level of transcription. In the first part of my thesis, the synthesis of photocaged 2'-deoxyribonucleosides triphosphates and their photolysis to unprotected 5-hydroxymethylated nucleotides is described. All prepared nucleoside triphosphates were good substrates for their enzymatic incorporation into DNA. Synthesized 5-(2-nitrobenzyloxy)methyl-2'-deoxyuridine-5'- monophosphate (dUNBMP) and DNA with one 5-(2-nitrobenzyloxy)methyl- modification in the sequence were used for the detailed kinetic studies of photocleavage reactions. In the second part of the thesis, the series of modified DNAs with specific sequences were prepared by primer extension (PEX) and/or polymerase chain reaction (PCR). A cleavage of prepared modified DNAs was studied by selected restriction endonucleases (REs). In all cases, the nitrobenzylated DNA fully resist the cleavage by REs. The deprotection/ photocleavage conditions for nitrobenzylated DNA were studied in the case of DNAs with positive restriction endonuclease digestion of hydroxymethylated DNA. The resulting photocleaved DNA was fully digested by REs, therefore 2-nitrobenzyl...