| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 94 === The R-loop structure, an RNA/DNA hybrid with a single-stranded DNA region, has been observed during transcription elongation. Recently, the formation of transcription-associated R-loop and the activation-induced cytidine deaminase (AID) have been demonstrated to be tightly associated with the recombination events around antibody maturation. In addition to serve as recombination substrates, R-loop might also present as activation signal for cellular stress response and as primer for the initiation of DNA replication. In this thesis, we took advantage of a bacterial model system to investigate the roles of DNA topoisomerases and recombination enzymes in the regulation of R-loop formation which is indicated by changes in the level of the AID-stimulated mutagenesis (ASM). Our results provided the first evidence that R-loop is indeed the targeting substrates for AID-induced mutagenesis. In addition, R-loop is also responsible for plasmid-mediated lethality and cellular filamentations in recBCsbcBC mutant background. Consistent with the proposed role for TopA in suppression of R-loop formation, our results demonstrated that topA mutation and over-expression of TopA increased and reduced the frequencies of ASM, respectively. Similarly, deficiency of another Type I topoisomerase, TopB, also resulted in higher level of ASM. On the other hand, the presence of DNA gyrase increased ASM, possibly through its R-loop promoting activity. Interestingly, another Type II topoisomerase, Topo IV, plays a suppressing role in the function of R-loop. As for the recombination enzymes, enzymes in the mismatch repair (MMR) and RecF pathways were required for ASM. Furthermore, the RecF pathway, by modulation of R-loop formation, contributes to plasmid-mediated lethality and cellular filamentations in recBCsbcBC mutant background.
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