| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 105 === R-loop, which is a cellular structure composing of an RNA/DNA hybrid and a displaced single-stranded DNA with two single and double-stranded junctions, could be a potent source causing genome instability. This genome instability has been suggested to be induced by the ability of R-loop to introduce DNA damage. Hence, cells have evolved various mechanisms to prevent excessive co-transcriptional R-loop formation such as RNase H enzyme to dissolve specifically the RNA/DNA hybrid. Many studies in prokaryotic cells have suggested that DNA damage initiates/activates the “SOS response” for boosting DNA repair capacity in order to maintain genomic integrity; however, with expression of repair polymerase and subsequent elevated level of error-prone replication, SOS response might result in the antibiotic resistance. In our study, correlated with our lab’s previous results, we first found that the cellular R-loop level in the topA10 mutant is higher than that of wild-type strain. In agreement, overexpression of RNase H can effectively suppress the amount of R-loop in cells, which is evidenced by direct detection with the advent of S9.6 antibodies in vitro and the Sγ3 (containing R-loop-prone sequence)-plasmid-mediated lethality in cells. Notably, our results revealed that R-loop can not only introduce the degradation of LexA protein but also be responsible for the phenotype of cellular filamentation, indicating a potential role of R-loop as a resource of SOS response and/or that R-loop formation leads to DNA damage. However, both the topA mutation and overexpression of functional RNase H in cells could restrict this mechanism. These data suggested a complicated regulation of SOS response that in addition to the negative regulatory role of R-loop formation and corresponding activation of SOS response, TopA also plays a direct role in activation of SOS response. Third, our results showed the first evidence that cells with higher cellular levels of R-loop have a reduced sensitivity to trimethoprim and quinolone antimicrobials (i.e. higher antibiotic resistance) and in agreement, reducing the cellular levels of R-loop by plasmid-mediated expression of RNase H in cells can then increase the sensitivity to these antimicrobials (i.e. lower antibiotic resistance). These observations suggested novel notions, those are supported by literature reports, that R-loop can activate SOS response thus leading to the subsequent antibiotic resistance. In addition, possibly through negatively regulation of the R-loop level inside a bacterial cell, TopA and RNase H suppress SOS response and antibiotic resistance. Last, our previous studies also found that in addition to topoisomerase I, nucleoid-associated proteins (NAPs) could also effectively influence the DNA topology. With the postulation that the factors involved in changing the topology and structure of DNA may participate in the regulation of R-loop formation, we further explored the potential role of NAPs such as HU and IHF in R-loop formation by the supercoiling assay. In sum, our results implicated that R-loop plays as a potential role in activating DNA damage-related SOS response and subsequently introducing the SOS response-mediated antimicrobial resistance. Although the TopA deficiency caused an elevated level of R-loop in cells, it is noted that our and other results also suggest TopA is also critically involved in the activation of SOS response. Thus, in the presence of TopA mutant, excess R-loop formation cannot activate SOS response and thus conferring hypersensitivity of topA mutant cells to antibiotics. Furthermore, factors involved in organization of nucleoid DNA also participate in the regulation of R-loop formation, suggesting that they may contribute to maintenance of genome integrity and play a potential role underlying antibiotic resistance. The importance of our findings to the emergent antibiotic resistance needs further investigation.
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