Le génome de tous les organismes vivants est constamment menacé par de nombreux agents qui causent des dommages à l'ADN. Lorsque la fourche de réplication rencontre une lésion de l'ADN non réparée, deux voies de tolérance aux dommages de l'ADN sont possibles : la voie de synthèse translésionnelle (TransLesion Synthesis - TLS) potentiellement mutagène et les voies fidèles de contournement des lésions (Damage Avoidance - DA) qui utilisent l'information du brin complémentaire non endommagé. L’équilibre entre ces deux voies de tolérance fidèle et mutagène est un paramètre essentiel puisqu’il permet de définir le niveau de mutagénèse lors de la réplication d'un ADN endommagé. Afin d’étudier in vivo ces différentes voies de tolérance ainsi que leur équilibre, le laboratoire a développé un système qui permet l’introduction d’une lésion unique sur le chromosome de la bactérie Escherichia coli. Il a montré que cet équilibre est contrôlé par le niveau d'expression des ADN polymérases translésionnelles, ainsi que par la capacité de la cellule à faire de la recombinaison homologue. Au cours de cette thèse, nous montrons que cet équilibre est également régulé par des contraintes structurales au niveau de la fourche de réplication. Nous montrons que la proximité de deux lésions sur des brins opposés entraine une forte inhibition de la voie fidèle DA due à un chevauchement des régions d’ADN simple brins générées en aval des lésions. Cette inhibition conduit alors à une augmentation de la TLS, indépendamment de la réponse SOS. Ces données révèlent ainsi que la proximité des lésions de l'ADN est un facteur essentiel dans l’équilibre des voies de tolérance, pouvant favoriser la mutagénèse. / The genome of all living organisms is constantly injured by several agents that cause DNA damages. When the replication fork encounters an unrepaired DNA lesion, two DNA damage tolerance pathways are possible : the potentially mutagenic Translesion Synthesis (TLS) pathway and the error-free Damage Avoidance (DA) pathways that use the information of the undamaged complementary strand. The balance between these error-free and error-prone tolerance pathways is an essential parameter since it defines the level of mutagenesis during replication of a damaged DNA. In order to study these different pathways of tolerance and their balance in vivo, the laboratory has developed a genetic system that allows the introduction of a single lesion on the chromosome of Escherichia coli. They showed that the balance is controlled by the level of expression of the TLS polymerases, as well as the capacity of the cell to perform homologous recombination. In this thesis, we show that this balance is also modulated by structural constraints at the level of the replication fork. We show that the proximity of two lesions on opposite strands results in a strong inhibition of DA due to an overlap of the single stranded DNA regions generated downstream of the lesions. This inhibition leads to an increase in TLS independently of the SOS response. These data reveal that the proximity of DNA lesions is an essential factor in the balance of DDT pathways, favoring mutagenesis.

We demonstrate that hydrogen production can be increased by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be further increased in the chemically-mutagenized strain by targeted gene deletion and overexpression of genes related to formate metabolism. Chemical mutagenesis of Escherichia coli BW25113 hyaB hybC hycE::kan/pBS(Kan)-HycE to form strain 3/86 resulted in 109 +/- 0.5- fold more hydrogen; 3/86 lacks functional hydrogen uptake hydrogenases 1 and 2, has hydrogenproducing hydrogenase 3 inactivated from the chromosome, and has constitutively active hydrogenase 3 based on expression of the large subunit of hydrogenase 3 from a high copy number plasmid. Deleting fdoG, which encodes formate dehydrogenase O, (that diverts formate from hydrogen), from chemical mutagen 3/86 increased hydrogen production 188 +/- 0.50-fold (relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of formate hydrogen lyase (FHL), increased hydrogen production 232 +/- 0.50-fold. Deleting both fdoG and hycA increased hydrogen production 257 +/- 0.50-fold, and overexpressing fhlA along with the fdoG hycA mutations increased hydrogen 308 +/- 0.52-fold. Whole-transcriptome analysis of chemical mutagen 3/86 revealed 89 genes were induced and 31 genes were repressed. In an effort to identify chromosomal mutations in chemical mutagen 3/86, we performed comparative genome sequencing and identified two chromosomal loci with mutations in coding regions of ftnA and yebJ; however, neither gene was related to the increased hydrogen production as determined by the close vial (short) hydrogen assay. In addition, transposon mutagenesis, which is one of the most efficient strategies for creating random mutations in the genomic DNA, was performed in two different strains: E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA and E. coli MG1655 to identify beneficial mutations for hydrogen production. As a result of screening 461 E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants and 1000 E. coli MG1655 transformants, three interesting mutations have been discovered in E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants (gpsA, dipZ, glgP) and 1 beneficial mutation in E. coli MG1655 transformants (malT). When any of these genes gpsA, dipZ, or glgP is disrupted by Tn5 insertion, hydrogen production decreases 17, 3 and 8-fold, respectively. Additionally, when malT gene is disrupted by Tn5 insertion, hydrogen increases 3.4-fold.

The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.

The role of specific amino acid residues in β-ketoadipate succinyl-coenzyme A transferase II from Acinetobacter calcoaceticus was investigated. A 1412 base pair BamiHI-EcoRI fragment carrying the catIJ genes was amplified by polymerase chain reaction and inserted into pUCl9 to generate the plasmid pCATEl9. Escherichia coli DH5α (pCATEl9) carrying only the catlJ genes expressed 3-fold higher enzyme activity than the parent strain. Two mutants were constructed by site directed mutagenesis so that glutamate was replaced by a glutamine at positions Gln155 and Gln193 in the ß subunit of the primary amino acid sequence of the CoA transferase. Both mutants produced transferase that was catalytically active suggesting that Glu155 and Glu193 do not participate directly in catalysis.

Call number: LD2668 .T4 1966 H749 / Master of Science

Call number: LD2668 .T4 1966 S127 / Master of Science

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.