<p>5-azacytidine and its derivatives are cytidine analogs used for leukemia chemotherapy. The primary effect of 5-azacytidine is the prohibition of cytosine methylation, which results in covalent DNA-methyltransferase crosslinks at cytosine methylation sites. These DNA-protein crosslinks have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms of DNA damage and the repair pathways of DNA-protein crosslinks have not been elucidated. </p><p>We used 2-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of 5-azacytidine. 2-dimensional gel analysis revealed the accumulation of specific bubble- and Y-molecules, dependent on overproduction of the cytosine methyltransferase EcoRII and treatment with 5-azacytidine. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble- and Y-molecules. These results imply that 5-azacytidine-induced DNA-protein crosslinks block DNA replication in vivo. RecA-dependent X-structures were also observed after 5-azacytidine treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles after 5-azacytidine treatment. These results suggest that replication can switch from theta to rolling circle mode after a replication fork is stalled by a DNA-methyltransferase crosslink. The simplest model for the conversion of theta to rolling-circle mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity. </p><p>In addition, we performed a transposon mutagenesis screen and found that mutants defective in the tmRNA translational quality control system are hypersensitive to 5-azacytidine. The hypersensitivity of these mutants requires expression of active methyltransferase, indicating that hypersensitivity is dependent on DNA-methyltransferase crosslink formation. Furthermore, the tmRNA pathway is activated upon 5-azacytidine treatment in cells expressing methyltransferase, resulting in increased SsrA tagging of cellular proteins. These results support a "chain-reaction" model, in which transcription complexes blocked by 5-azacytidine-induced DNA-protein crosslinks result in ribosomes stalling on the attached nascent transcripts, and the tmRNA pathway is invoked for cleaning up the resulting pile-ups. In support of this model, an ssrA mutant is also hypersensitive to antibiotic streptolydigin, which blocks RNA polymerase elongation. These results reveal a novel role for the tmRNA system in clearance of coupled transcription/translation complexes in which RNA polymerase has become blocked.</p> / Dissertation

La méthylation de l’adénine en position 58 des ARNt (m1A58) est présente dans les trois domaines de vie et joue un rôle crucial chez plusieurs organismes. Sa formation est catalysée par la méthyltransférase SAM-dépendante TrmI. Alors que chez les eucaryotes et les bactéries, TrmI est site-spécifique pour l’adénine en position 58, chez l’archée Pyrococcus abyssi, TrmI est région-spécifique puisqu’elle catalyse également la méthylation de l’adénine en position 57. Nous nous sommes intéressés à cette enzyme, PabTrmI, pour comprendre cette différence de spécificité par rapport à ses homologues eucaryotes et bactériens.La structure cristallographique de l'enzyme, en complexe avec son cofacteur SAM ainsi qu’avec le produit de la réaction, la SAH, nous a conduit à construire différents mutants de la protéine et de son substrat ARNt. Nous avons ainsi montré que His78, située à l’entrée du site actif, est mobile et est importante pour l’efficacité catalytique de PabTrmI. L’analyse des positions de méthylation par spectrométrie de masse, simple et en tandem, montre qu’une partie de la région-spécificité de l’enzyme pour certains ARNt de P. abyssi, est liée à la présence de trois adénines consécutives, PabTrmI ne méthylant que la première adénine d’une séquence AA.En vue d’étudier les cinétiques rapides du mécanisme de retournement de la base de l’ARNt par l’enzyme région-spécifique PabTrmI et l’enzyme bactérienne site-spécifique TthTrmI de T. thermophilus, nous avons vérifié dans un premier temps, par spectrométrie de masse, qu’un mini-ARNt, constitué de la tige acceptrice et de la tige-boucle T, est substrat de TrmI. / The methylation of adenine 58 in the T-loop of tRNAs (m1A58) is a modification present in all three domains of life. An important biological role for m1A58 has been demonstrated in different organisms. Its formation is catalyzed by the SAM-dependent methyltransferase TrmI. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi is region-specific, since it catalyzes the formation of m1A also at the adjacent position 57. A structural and biochemical study of this enzyme, PabTrmI, was undertaken to shed light on the origin of the multisite recognition mechanism.First, we determined the crystal structure of PabTrmI in complex with its cofactor SAM and the reaction product SAH. These structures enabled us to construct both protein and tRNA mutants. We show that His78, which lies near the active site, is mobile and important for catalytic efficiency of PabTrmI. The analysis of the méthylation positions, by mass spectrometry, shows that at least part of the enzyme region-specificity for several P. abyssi tRNAs, is related to the presence of three consecutive adenines, with the enzyme modifying the first adenine of an AA sequence.To investigate the mechanism of tRNA base flipping by the region-specific enzyme PabTrmI and the site-specific bacterial enzyme TthTrmI of T. thermophilus, we checked, as a first step, by mass spectrometry, that a mini-tRNA, formed from the acceptor stem and the T-arm, was substrate of TrmI.