臺灣博碩士論文加值系統

細胞中，被認為含有RNA和DNA互補配對及單股形式存在的非模板DNA結構被稱為是R-loop，此結構可能會引起DNA受損而造成基因不穩定性。因此，細胞演化出許多機制像是RNase H來調控R-loop在細胞中的量。此外，有研究顯示在原核生物中，DNA受損後會緊急啟動「SOS反應」來修復DNA損傷部位來維持基因體的完整性；然而由於錯誤傾向修復聚合酶也會在SOS反應中被誘發，造成修復後的基因突變，進而可能導致細菌對於抗生素產生抗性的原因之一。在我們的研究，我們首先利用S9.6單株抗體以及Sγ3（含好發R-loop的DNA序列）質體造成的死亡現象來探討細菌中R-loop的含量。我們發現：和實驗室之前結果吻合，在topA10突變菌株中，R-loop含量比野生型來的多；而在細菌體內額外表達RNase H時，細菌體內的R-loop含量則會比額外表達突變的RNase H來的少。接著，我們發現R-loop可以造成LexA蛋白的分解以及造成「細胞長絲化（cellular filamentation）」的現象，暗示著R-loop可能為SOS反應上游的來源之一或是R-loop可能會引起DNA損傷；然而，有趣的是，在topA10菌株中，我們卻沒有看到這樣的現象，意指TopA對SOS反應的活化也扮演重要角色。另外，我們在本論文中呈現了第一個證據表示細菌中擁有比較多的R-loop含量會對於trimethoprim以及奎寧類抗生素有比較低的敏感性（即提高抗生素抗性），而相同地，當我們在細菌中額外表現RNase H來減少細胞中的R-loop含量時，就會增加細菌對於trimethoprim以及奎寧類抗生素的敏感性（即降低抗生素抗性）。以上結果闡明兩個假說：R-loop可以活化SOS反應，進而導致抗生素的抗性；另外，TopA和RNase H藉由負向調控R-loop的生成，壓抑SOS反應的產生（但，TopA也對SOS反應的活化扮演重要角色），進而可能調節細菌對抗生素的抗性。最後，由於R-loop的形成和DNA拓樸結構有關，而我們先前實驗室發現不只拓樸異構酶，類核體相關蛋白也會參與在調控DNA拓樸結構中，因次我們也利用了含有氯奎寧的膠體電泳來觀察含不同類和體相關蛋白的菌株菌株中報告質體之DNA超螺旋變化來看類核體相關蛋白，像是HU以及IHF，在R-loop生成中所扮演的角色。綜合我們以上的結果，我們發現R-loop可以作為SOS反應的啟動來源，並第一次提供實驗證據表示R-loop也可能造成SOS反應衍生、引起的抗生素抗性。綜合先前我們實驗室以及本論文結果，我們認為類核體相關蛋白也可能參與在R-loop生成中，這也暗示著這些蛋白也可能在維持基因完整性和R-loop造成的抗生素抗性中扮演一些角色。有趣的是，細胞長絲化的現象和臨床上發現抗抗生素的大腸桿菌之產生及大腸桿菌的輻射抗性有關，因此我們相信此論文的發現對目前未解決的抗生素抗性問題提供了一個解答方向，非常值得進一步研究。

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.

致謝 i
中文摘要 ii
ABSTRACT iv
CONTENTS vi
INTRODUCTION 1
1. Transcription 1
1.1. Transcription as a source of genome instability 2
1.1.1. Transcription-associated mutagenesis and co-transcriptional R-loop 2
1.1.2. Transcription-associated recombination and co-transcriptional R-loop 3
2. R-loop 4
2.1. Formation of R-loop 5
2.1.1. Two proposed models for R-loop formation 5
2.2. Biological functions of R-loop 7
2.2.1. The role of R-loop in the initiaion of DNA replication 7
2.2.2. R-loop-mediated Ig class-switch recombination in B cells 8
2.2.3. R-loop and cell growth inhibition 8
2.2.4. R-loop-mediated genome instability 9
2.3. Regulation of R-loop 10
2.3.1. RNase H enzymes in restricting of R-loop formation 11
2.3.2. The role of topoisomerases and transcription-dependent supercoiling in the regulation of R-loop formation 11
2.3.3. Co-transcriptional RNA-processing and RNA-export factors in preventing the R-loop formation 12
2.3.4. Other factors involved in the regulation of R-loop formation 13
3. SOS response 14
3.1. SOS genes and their induction order 15
3.2. Origin of the SOS-inducing single-stranded DNA (ssDNA) 16
3.3. SOS response and bacterial antibiotic resistance 16
4. Antibiotics 17
4.1. Types of antibiotic 18
4.2. Antibiotic-induced SOS response 19
4.2.1. Sub-MICs antibiotics and SOS response 19
5. DNA topoisomerases 20
5.1. General features of type I DNA topoisomerases 21
5.2. General features of type II DNA topoisomerase 21
5.3. Physiology role of topoisomerases in Escherichia coli 22
6. Nucleoid-associated proteins (NAPs) 23
6.1. NAPs and DNA supercoiling 23
SPECIFIC AIMS 25
MATERIALS AND METHODS 26
Escherichia coli strains 26
Plasmids 26
Media and growth conditions 27
Preparation of competent cells 27
Transformation 28
S9.6 antibody production 28
S9.6 antibody purification and dialysis 28
Dot blot analysis for R-loop detection 29
Spotting assay 30
LexA protein analysis 30
Fluoresence microscopy used for cellular filamentation 30
Luciferase assay 31
Assays for activity of antimicrobial agents 32
Plasmid extraction for supercoiling analysis 32
Plasmid topoisomer analysis 32
Statistic analysis 33
RESULTS 34
Effects of RNase A and/or RNase H treatments on S9.6 signaling in the dot-blotting analysis. 35
Overexpression of RNase H can reduce the cellular levels of R-loop and topA10 mutation in bacterial cells have higher cellular amount of R-loop. 36
Overexpression of RNase H can reduce the plasmid-mediated lethality caused by transcription through Sγ3 region. 36
Overexpression of RNase H can reduce the cellular filamentation and LexA degradation caused by transcription through Sγ3 region. 37
Overexpression of RNase H can reduce the SOS response induction mediated by mitomycin C. 39
Plasmid-mediated lethality caused by transcription through Sγ3 region increases in topA10 mutant. 39
Deficient of topoisomerase I restricts the cellular filamentation and LexA degradation caused by transcription through Sγ3 region. 40
Topoisomerase I function is required for SOS response induction mediated by mitomycin C. 41
Determine the cell growth of strains with Ts DNA gyrase and topA deletion transformed with distinct plasmids at different temperature. 41
Plasmid-mediated lethality caused by transcription through Sγ3 region in strains with gyrBts at 37℃. 42
Determine the optimal condition for investigate the effect of topoisomerase deletion on SOS response initiation. 43
Deletion of topoisomerase I in strains show more severe plasmid-mediated lethality than isogenic WT strain with gyrBts and overexpression of RNase H can ameliorate the plasmid-mediated lethality in WT at 30℃. 44
Overexpression of RNase H and deficient of topoisomerase I both restrict the cellular filamentation and LexA degradation caused by transcription through Sγ3 region in strains with gyrBts at 30℃. 44
Effect of topA10 mutation on growth inhibition by antimicrobials 46
Overexpression of RNase H can slightly reduce the growth inhibition by antimicrobials. 46
Determine differential superhelical density in the NAP-deleted strains 47
Superhelical density of DPB923 (topA10) is similar to YK2741 (ΔhupA ΔhupB ΔhimA) 47
DISCUSSION 49
Filamentous growth may not only result from sulA-dependent mechanism 49
Filamentous growth is associated with antibiotic resistance and radiation resistance 50
Deficiency and deletion of topoisomerase I lead to restrict the SOS response initiation 51
Growth inhibition by antimicrobials in strain with deficiency of topoisomerase I, which restricts the SOS response initiation. 52
Role of R-loop in SOS-mediated antibiotic resistance 53
Overexpression of RNase H can ameliorate the luciferase activity mediated by mitomycin C 54
Higher superhelical density in double mutant strains YK1340 (ΔhupA ΔhupB) 55
REFERENCES 57
TABLES 70
Table I. Escherichia coli strains used in this study 70
Table II. Plasmids and constructions used in this study 72
Table III. Survival rate of DPB924 (topA+) and DPB923 (topA10) with pPTac-Sγ3-F on IPTG-containing plate 73
Table IV. Survival rate of RFM445 (gyrBts) and RFM475 (ΔtopA gyrBts) with pPTac-Sγ3-F on IPTG-containing plate at 37℃ 74
Table V. Survival rate of RFM445 (gyrBts) and RFM475 (ΔtopA gyrBts) with pPTac-Sγ3-F on IPTG-containing plate at 30℃ 75
Table VI. Effect of topA10 mutation on MICs 76
Table VII. Effect of ectopic expression of RNase H in DPB924 (WT) on MICs 77
Table VIII. Effect of ectopic expression of RNase H in DPB923 (topA10) on MICs 78
Table VIIII. The postulation of cellular levels of R-loop in RFM445 and RFM475 among distinct temperature. 79
FIGURES 80
Figure 1. The signals detected by the Dot-blotting with S9.6 antibodies represent the amounts of RNA:DNA hybrid, possible of R-loop. 80
Figure 2. The concentrations of RNase A between 10 µg/ml to 50 µg/ml can degrade the single-strand RNA completely without affecting the Dot-blotting signals of S9.6 antibodies. 82
Figure 3. Ectopic expression of functional RNase H can reduce the cellular level of RNA/DNA hybrids in DPB924 wild-type cells. 83
Figure 4. The mutation of topA10 shows higher cellular levels of RNA:DNA hybrids than WT. 84
Figure 5. Cell growth defect and plasmid loss caused by transcription through a plasmid-borne Sγ3 region in its physiological orientation can be ameliorated by ectopic expression of RNase H in DPB924 (WT). 86
Figure 6. Cellular filamentation induced by transcription through a plasmid-borne Sγ3 region in its physiological orientation can be ameliorated by RNase H in DPB924 (WT). 88
Figure 7. IPTG-induced SOS response-associated LexA protein degradation is more severe at 60 min. 89
Figure 8. IPTG-induced SOS response-associated LexA protein degradation can be alleviated by ectopic overexpressing of RNase H. 90
Figure 9. Overexpression of RNase H can diminish the SOS induction mediated by mitomycin C in DPB924 (WT). 92
Figure 10. The lower activity of topoisomerase I can lead to higher Sγ3-mideated lethality. 94
Figure 11. The lower activity of Topoisomerase I can restrict the Sγ3-mediated cellular fialmentation. 96
Figure 12. The lower activity of topoisomerase I can restrict the degradation of SOS response-associated LexA protein. 98
Figure 13. Topoisomerase I function plays a crucial role for SOS induction mediated by mitomycin C. 100
Figure 14. To demonstrate the growth characteristic of RFM475 (ΔtopA gyrBts) transformed with distinct plasmids between 37℃ and 28℃. 101
Figure 15. The Sγ3-mediated lethality in cells with gryBts can be diminished at non-permissive temperature and the lethality cannot be rescued by ectopic expression of RNase H. 103
Figure 16. The growth of cells with gyrBts mutant and ΔtopA gyrBts double mutants at the permissive, semi-permissive and non-permissive temperature. 104
Figure 17. Ectopic expression of RNase H and topoisomerase I can partially and fully rescue the topA deletion in RFM475, respectively at 28℃ after 24 hours growth. 107
Figure 18. The Sγ3-mediated lethality of RFM445 (gyrBts) and RFM475 (ΔtopA gyrBts) is correlated with DPB924 (WT)and DPB923 (topA10) at permissive temperature. 109
Figure 19. Sγ3-mediated cellular filamentation can be restricted by deletion of topoisomerase I in transfromants with gyrB203ts and be ameliorated by ectopic expression of RNase H in RFM445 (gyrBts) at permissive temperature. 111
Figure 20. SOS damage response induced in cells with gyrB203ts transformed with pPtac-Sγ3-F at non-permissive temperature; however, not induced in RFM445 with gyrB203ts ΔtopA double mutant cells. 112
Figure 21. The mutation of topA10 results in more severe growth inhibition by antimicrobial. 113
Figure 22. Ectopic expression of RNase H can reduce the viability of DPB924 (WT) on Mueller-Hinton plate containing ciprofloxacin, norfloxacin and trimethoprim. 114
Figure 23. Distinct NAPs strains show different distribution of supercoiling level and YK2741 shows more negative level of superhelical density compared to other strainsk; besides, YK1340 shows less negative. 115
Figure 24. The superhelical density of both DPB923 and YK2741 are relatively more negative than WT. 116
Figure 25. The schematic representation for our working hypotheses, results and proposed model for regulation and biological functions of R-loop. 117

Adams, D. W., & Errington, J. (2009). Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nat Rev Microbiol, 7(9), 642-653.
Aguilera, A. (2002). The connection between transcription and genomic instability. Embo j, 21(3), 195-201.
Aguilera, A., & Garcia-Muse, T. (2012). R loops: from transcription byproducts to threats to genome stability. Mol Cell, 46(2), 115-124.
Aguilera, A., & Klein, H. L. (1990). HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene. Mol Cell Biol, 10(4), 1439-1451.
Aldridge, M., Facey, P., Francis, L., Bayliss, S., Del Sol, R., & Dyson, P. (2013). A novel bifunctional histone protein in Streptomyces: a candidate for structural coupling between DNA conformation and transcription during development and stress? Nucleic Acids Res, 41(9), 4813-4824.
Baaklini, I., Hraiky, C., Rallu, F., Tse-Dinh, Y. C., & Drolet, M. (2004). RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli. Mol Microbiol, 54(1), 198-211.
Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., . . . Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol, 2, 2006 0008.
Baharoglu, Z., Babosan, A., & Mazel, D. (2014). Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase. Nucleic Acids Res, 42(4), 2366-2379.
Baharoglu, Z., & Mazel, D. (2014). SOS, the formidable strategy of bacteria against aggressions. FEMS Microbiol Rev, 38(6), 1126-1145.
Beale, R. C., Petersen-Mahrt, S. K., Watt, I. N., Harris, R. S., Rada, C., & Neuberger, M. S. (2004). Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. J Mol Biol, 337(3), 585-596.
Bensaid, A., Almeida, A., Drlica, K., & Rouviere-Yaniv, J. (1996). Cross-talk between topoisomerase I and HU in Escherichia coli. J Mol Biol, 256(2), 292-300.
Bergerat, A., de Massy, B., Gadelle, D., Varoutas, P. C., Nicolas, A., & Forterre, P. (1997). An atypical topoisomerase II from Archaea with implications for meiotic recombination. Nature, 386(6623), 414-417.
Biek, D. P., & Cohen, S. N. (1989). Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF. J Bacteriol, 171(4), 2056-2065.
Boguslawski, S. J., Smith, D. E., Michalak, M. A., Mickelson, K. E., Yehle, C. O., Patterson, W. L., & Carrico, R. J. (1986). Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J Immunol Methods, 89(1), 123-130.
Broccoli, S., Rallu, F., Sanscartier, P., Cerritelli, S. M., Crouch, R. J., & Drolet, M. (2004). Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation. Mol Microbiol, 52(6), 1769-1779.
Burton, P., & Holland, I. B. (1983). Two pathways of division inhibition in UV-irradiated E. coli. Mol Gen Genet, 190(1), 128-132.
Champoux, J. J. (2001). DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem, 70, 369-413.
Chan, Y. A., Aristizabal, M. J., Lu, P. Y., Luo, Z., Hamza, A., Kobor, M. S., . . . Hieter, P. (2014). Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip. PLoS Genet, 10(4), e1004288.
Chaudhuri, J., & Alt, F. W. (2004). Class-switch recombination: interplay of transcription, DNA deamination and DNA repair. Nat Rev Immunol, 4(7), 541-552.
Chavez, S., & Aguilera, A. (1997). The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability. Genes Dev, 11(24), 3459-3470.
Chen, X., Yang, J. R., & Zhang, J. (2016). Nascent RNA folding mitigates transcription-associated mutagenesis. Genome Res, 26(1), 50-59.
Chen, X., & Zhang, J. (2014). Yeast mutation accumulation experiment supports elevated mutation rates at highly transcribed sites. Proc Natl Acad Sci U S A, 111(39), E4062.
Cirz, R. T., Chin, J. K., Andes, D. R., de Crecy-Lagard, V., Craig, W. A., & Romesberg, F. E. (2005). Inhibition of mutation and combating the evolution of antibiotic resistance. PLoS Biol, 3(6), e176.
Cirz, R. T., Jones, M. B., Gingles, N. A., Minogue, T. D., Jarrahi, B., Peterson, S. N., & Romesberg, F. E. (2007). Complete and SOS-mediated response of Staphylococcus aureus to the antibiotic ciprofloxacin. J Bacteriol, 189(2), 531-539.
Conticello, S. G. (2008). The AID/APOBEC family of nucleic acid mutators. Genome Biol, 9(6), 229.
Courcelle, J., Khodursky, A., Peter, B., Brown, P. O., & Hanawalt, P. C. (2001). Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics, 158(1), 41-64.
Daniels, G. A., & Lieber, M. R. (1995). RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination. Nucleic Acids Res, 23(24), 5006-5011.
Datta, A., & Jinks-Robertson, S. (1995). Association of increased spontaneous mutation rates with high levels of transcription in yeast. Science, 268(5217), 1616-1619.
de Vries, R. (2010). DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins. Biochimie, 92(12), 1715-1721.
DiGate, R. J., & Marians, K. J. (1989). Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III. J Biol Chem, 264(30), 17924-17930.
Dillon, S. C., & Dorman, C. J. (2010). Bacterial nucleoid-associated proteins, nucleoid structure and gene expression. Nat Rev Microbiol, 8(3), 185-195.
Dinardo, S., Voelkel, K. A., Sternglanz, R., Reynolds, A. E., & Wright, A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell, 31(1), 43-51.
Dorman, C. J. (2013a). Co-operative roles for DNA supercoiling and nucleoid-associated proteins in the regulation of bacterial transcription. Biochem Soc Trans, 41(2), 542-547.
Dorman, C. J. (2013b). Genome architecture and global gene regulation in bacteria: making progress towards a unified model? Nat Rev Microbiol, 11(5), 349-355.
Drlica, K. (1992). Control of bacterial DNA supercoiling. Mol Microbiol, 6(4), 425-433.
Drlica, K., Malik, M., Kerns, R. J., & Zhao, X. (2008). Quinolone-mediated bacterial death. Antimicrob Agents Chemother, 52(2), 385-392.
Drolet, M. (2006). Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology. Mol Microbiol, 59(3), 723-730.
Drolet, M., Bi, X., & Liu, L. F. (1994). Hypernegative supercoiling of the DNA template during transcription elongation in vitro. J Biol Chem, 269(3), 2068-2074.
Drolet, M., Broccoli, S., Rallu, F., Hraiky, C., Fortin, C., Masse, E., & Baaklini, I. (2003). The problem of hypernegative supercoiling and R-loop formation in transcription. Front Biosci, 8, d210-221.
Drolet, M., Phoenix, P., Menzel, R., Masse, E., Liu, L. F., & Crouch, R. J. (1995). Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. Proc Natl Acad Sci U S A, 92(8), 3526-3530.
El Hage, A., French, S. L., Beyer, A. L., & Tollervey, D. (2010). Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev, 24(14), 1546-1558.
El Hage, A., Webb, S., Kerr, A., & Tollervey, D. (2014). Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria. PLoS Genet, 10(10), e1004716.
Fleming, A. (1929). On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to their Use in the Isolation of B. influenzæ. British journal of experimental pathology, 10(3), 226-236.
Forterre, P. (2001). Genomics and early cellular evolution. The origin of the DNA world. Comptes Rendus de l''Académie des Sciences - Series III - Sciences de la Vie, 324(12), 1067-1076.
Fuchs, R. P., & Fujii, S. (2013). Translesion DNA synthesis and mutagenesis in prokaryotes. Cold Spring Harb Perspect Biol, 5(12), a012682.
Fujii, S., Isogawa, A., & Fuchs, R. P. (2006). RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis. Embo j, 25(24), 5754-5763.
Gan, W., Guan, Z., Liu, J., Gui, T., Shen, K., Manley, J. L., & Li, X. (2011). R-loop-mediated genomic instability is caused by impairment of replication fork progression. Genes Dev, 25(19), 2041-2056.
Garcia-Rubio, M. L., Perez-Calero, C., Barroso, S. I., Tumini, E., Herrera-Moyano, E., Rosado, I. V., & Aguilera, A. (2015). The Fanconi Anemia Pathway Protects Genome Integrity from R-loops. PLoS Genet, 11(11), e1005674.
Gavalda, S., Gallardo, M., Luna, R., & Aguilera, A. (2013). R-loop mediated transcription-associated recombination in trf4Delta mutants reveals new links between RNA surveillance and genome integrity. PLoS One, 8(6), e65541.
Gowrishankar, J., & Harinarayanan, R. (2004). Why is transcription coupled to translation in bacteria? Mol Microbiol, 54(3), 598-603.
Groh, M., & Gromak, N. (2014). Out of balance: R-loops in human disease. PLoS Genet, 10(9), e1004630.
Groh, M., Silva, L. M., & Gromak, N. (2014). Mechanisms of transcriptional dysregulation in repeat expansion disorders. Biochem Soc Trans, 42(4), 1123-1128.
Guerin, E., Cambray, G., Sanchez-Alberola, N., Campoy, S., Erill, I., Da Re, S., . . . Mazel, D. (2009). The SOS response controls integron recombination. Science, 324(5930), 1034.
Gullberg, E., Cao, S., Berg, O. G., Ilback, C., Sandegren, L., Hughes, D., & Andersson, D. I. (2011). Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathog, 7(7), e1002158.
Gutierrez, A., Laureti, L., Crussard, S., Abida, H., Rodriguez-Rojas, A., Blazquez, J., . . . Matic, I. (2013). beta-Lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity. Nat Commun, 4, 1610.
Hamperl, S., & Cimprich, K. A. (2014). The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA Repair (Amst), 19, 84-94.
Hanawalt, P. C., & Spivak, G. (2008). Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol, 9(12), 958-970.
Herman, R. K., & Dworkin, N. B. (1971). Effect of gene induction on the rate of mutagenesis by ICR-191 in Escherichia coli. J Bacteriol, 106(2), 543-550.
Hong, X., Cadwell, G. W., & Kogoma, T. (1995). Escherichia coli RecG and RecA proteins in R-loop formation. Embo j, 14(10), 2385-2392.
Huang, F. T., Yu, K., Balter, B. B., Selsing, E., Oruc, Z., Khamlichi, A. A., . . . Lieber, M. R. (2007). Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region. Mol Cell Biol, 27(16), 5921-5932.
Huang, F. T., Yu, K., Hsieh, C. L., & Lieber, M. R. (2006). Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination. Proc Natl Acad Sci U S A, 103(13), 5030-5035.
Huertas, P., & Aguilera, A. (2003). Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol Cell, 12(3), 711-721.
Huisman, O., D''Ari, R., & George, J. (1980). Inducible sfi dependent division inhibition in Escherichia coli. Mol Gen Genet, 177(4), 629-636.
Itoh, T., & Tomizawa, J. (1980). Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A, 77(5), 2450-2454.
Janion, C. (2001). Some aspects of the SOS response system--a critical survey. Acta Biochim Pol, 48(3), 599-610.
Jena, N. R. (2012). DNA damage by reactive species: Mechanisms, mutation and repair. J Biosci, 37(3), 503-517.
Justice, S. S., Hunstad, D. A., Cegelski, L., & Hultgren, S. J. (2008). Morphological plasticity as a bacterial survival strategy. Nat Rev Microbiol, 6(2), 162-168.
Justice, S. S., Hunstad, D. A., Seed, P. C., & Hultgren, S. J. (2006). Filamentation by Escherichia coli subverts innate defenses during urinary tract infection. Proc Natl Acad Sci U S A, 103(52), 19884-19889.
Kano, Y., & Imamoto, F. (1990). Requirement of integration host factor (IHF) for growth of Escherichia coli deficient in HU protein. Gene, 89(1), 133-137.
Kasahara, M., Clikeman, J. A., Bates, D. B., & Kogoma, T. (2000). RecA protein-dependent R-loop formation in vitro. Genes Dev, 14(3), 360-365.
Kato, T., & Kondo, S. (1970). Genetic and molecular characteristics of X-ray-sensitive mutants of Escherichia coli defective in repair synthesis. J Bacteriol, 104(2), 871-881.
Keil, R. L., & Roeder, G. S. (1984). Cis-acting, recombination-stimulating activity in a fragment of the ribosomal DNA of S. cerevisiae. Cell, 39(2 Pt 1), 377-386.
Kelley, W. L. (2006). Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon. Mol Microbiol, 62(5), 1228-1238.
Keskin, H., Shen, Y., Huang, F., Patel, M., Yang, T., Ashley, K., . . . Storici, F. (2014). Transcript-RNA-templated DNA recombination and repair. Nature, 515(7527), 436-439.
Kjeldsen, T. S., Sommer, M. O., & Olsen, J. E. (2015). Extended spectrum beta-lactamase-producing Escherichia coli forms filaments as an initial response to cefotaxime treatment. BMC Microbiol, 15, 63.
Kogoma, T. (1997). Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription. Microbiol Mol Biol Rev, 61(2), 212-238.
Kohanski, M. A., DePristo, M. A., & Collins, J. J. (2010). Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol Cell, 37(3), 311-320.
Kohanski, M. A., Dwyer, D. J., & Collins, J. J. (2010). How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol, 8(6), 423-435.
Lal, A., Dhar, A., Trostel, A., Kouzine, F., Seshasayee, A. S. N., & Adhya, S. (2016). Genome scale patterns of supercoiling in a bacterial chromosome. Nature Communications, 7, 11055.
Lam, S. J., O''Brien-Simpson, N. M., Pantarat, N., Sulistio, A., Wong, E. H., Chen, Y. Y., . . . Qiao, G. G. (2016). Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers. Nat Microbiol, 1(11), 16162.
Lee, T. I., & Young, R. A. (2013). Transcriptional regulation and its misregulation in disease. Cell, 152(6), 1237-1251.
Leela, J. K., Syeda, A. H., Anupama, K., & Gowrishankar, J. (2013). Rho-dependent transcription termination is essential to prevent excessive genome-wide R-loops in Escherichia coli. Proc Natl Acad Sci U S A, 110(1), 258-263.
Li, X., & Manley, J. L. (2005). Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell, 122(3), 365-378.
Li, X., & Manley, J. L. (2006). Cotranscriptional processes and their influence on genome stability. Genes Dev, 20(14), 1838-1847.
Lind, P. A., & Andersson, D. I. (2008). Whole-genome mutational biases in bacteria. Proc Natl Acad Sci U S A, 105(46), 17878-17883.
Liu, I. F., Sutherland, J. H., Cheng, B., & Tse-Dinh, Y. C. (2011). Topoisomerase I function during Escherichia coli response to antibiotics and stress enhances cell killing from stabilization of its cleavage complex. J Antimicrob Chemother, 66(7), 1518-1524.
Liu, L. F., & Wang, J. C. (1987). Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A, 84(20), 7024-7027.
Loomis, E. W., Sanz, L. A., Chedin, F., & Hagerman, P. J. (2014). Transcription-associated R-loop formation across the human FMR1 CGG-repeat region. PLoS Genet, 10(4), e1004294.
Lopez, C. R., Yang, S., Deibler, R. W., Ray, S. A., Pennington, J. M., Digate, R. J., . . . Zechiedrich, E. L. (2005). A role for topoisomerase III in a recombination pathway alternative to RuvABC. Mol Microbiol, 58(1), 80-101.
Manis, J. P., Tian, M., & Alt, F. W. (2002). Mechanism and control of class-switch recombination. Trends Immunol, 23(1), 31-39.
Masse, E., & Drolet, M. (1999a). Escherichia coli DNA topoisomerase I inhibits R-loop formation by relaxing transcription-induced negative supercoiling. J Biol Chem, 274(23), 16659-16664.
Masse, E., & Drolet, M. (1999b). R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition. J Mol Biol, 294(2), 321-332.
Masse, E., Phoenix, P., & Drolet, M. (1997). DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli. J Biol Chem, 272(19), 12816-12823.
Mesak, L. R., & Davies, J. (2009). Phenotypic changes in ciprofloxacin-resistant Staphylococcus aureus. Res Microbiol, 160(10), 785-791.
Mesak, L. R., Miao, V., & Davies, J. (2008). Effects of subinhibitory concentrations of antibiotics on SOS and DNA repair gene expression in Staphylococcus aureus. Antimicrob Agents Chemother, 52(9), 3394-3397.
Michel, B. (2005). After 30 years of study, the bacterial SOS response still surprises us. PLoS Biol, 3(7), e255.
Miller, C., Thomsen, L. E., Gaggero, C., Mosseri, R., Ingmer, H., & Cohen, S. N. (2004). SOS response induction by beta-lactams and bacterial defense against antibiotic lethality. Science, 305(5690), 1629-1631.
Mischo, H. E., Gomez-Gonzalez, B., Grzechnik, P., Rondon, A. G., Wei, W., Steinmetz, L., . . . Proudfoot, N. J. (2011). Yeast Sen1 helicase protects the genome from transcription-associated instability. Mol Cell, 41(1), 21-32.
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., & Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell, 102(5), 553-563.
Park, C., Qian, W., & Zhang, J. (2012). Genomic evidence for elevated mutation rates in highly expressed genes. EMBO Rep, 13(12), 1123-1129.
Pennington, J. M., & Rosenberg, S. M. (2007). Spontaneous DNA breakage in single living Escherichia coli cells. Nat Genet, 39(6), 797-802.
Petersen-Mahrt, S. K., Harris, R. S., & Neuberger, M. S. (2002). AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature, 418(6893), 99-104.
Phillips, D. D., Garboczi, D. N., Singh, K., Hu, Z., Leppla, S. H., & Leysath, C. E. (2013). The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6. J Mol Recognit, 26(8), 376-381.
Phoenix, P., Raymond, M. A., Masse, E., & Drolet, M. (1997). Roles of DNA topoisomerases in the regulation of R-loop formation in vitro. J Biol Chem, 272(3), 1473-1479.
Piddock, L. J. (2012). The crisis of no new antibiotics--what is the way forward? Lancet Infect Dis, 12(3), 249-253.
Pohjoismaki, J. L., Holmes, J. B., Wood, S. R., Yang, M. Y., Yasukawa, T., Reyes, A., . . . Holt, I. J. (2010). Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol, 397(5), 1144-1155.
Pohlhaus, J. R., & Kreuzer, K. N. (2005). Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo. Mol Microbiol, 56(6), 1416-1429.
Pruss, G. J., & Drlica, K. (1986). Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling. Proc Natl Acad Sci U S A, 83(23), 8952-8956.
Radman, M. (1975). SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. Basic Life Sci, 5A, 355-367.
Richardson, J. P. (2002). Rho-dependent termination and ATPases in transcript termination. Biochim Biophys Acta, 1577(2), 251-260.
Richardson, J. P. (2003). Loading Rho to terminate transcription. Cell, 114(2), 157-159.
Roberts, R. W., & Crothers, D. M. (1992). Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science, 258(5087), 1463-1466.
Rudolph, C. J., Upton, A. L., Briggs, G. S., & Lloyd, R. G. (2010). Is RecG a general guardian of the bacterial genome? DNA Repair (Amst), 9(3), 210-223.
Sassanfar, M., & Roberts, J. W. (1990). Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J Mol Biol, 212(1), 79-96.
Savic, D. J., & Kanazir, D. T. (1972). The effect of a histidine operator-constitutive mutation on UV-induced mutability within the histidine operon of Salmonella typhimurium. Mol Gen Genet, 118(1), 45-50.
Sawitzke, J. A., & Austin, S. (2000). Suppression of chromosome segregation defects of Escherichia coli muk mutants by mutations in topoisomerase I. Proc Natl Acad Sci U S A, 97(4), 1671-1676.
Schneider, R., Travers, A., Kutateladze, T., & Muskhelishvili, G. (1999). A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli. Mol Microbiol, 34(5), 953-964.
Selzer, G., & Tomizawa, J. I. (1982). Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication. Proc Natl Acad Sci U S A, 79(23), 7082-7086.
Skourti-Stathaki, K., & Proudfoot, N. J. (2014). A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev, 28(13), 1384-1396.
Skourti-Stathaki, K., Proudfoot, N. J., & Gromak, N. (2011). Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol Cell, 42(6), 794-805.
Sollier, J., & Cimprich, K. A. (2015). Breaking bad: R-loops and genome integrity. Trends Cell Biol, 25(9), 514-522.
Swiercz, J. P., Nanji, T., Gloyd, M., Guarne, A., & Elliot, M. A. (2013). A novel nucleoid-associated protein specific to the actinobacteria. Nucleic Acids Res, 41(7), 4171-4184.
Tanaka, H., Yasuzawa, K., Kohno, K., Goshima, N., Kano, Y., Saiki, T., & Imamoto, F. (1995). Role of HU proteins in forming and constraining supercoils of chromosomal DNA in Escherichia coli. Mol Gen Genet, 248(5), 518-526.
Thomas, M., White, R. L., & Davis, R. W. (1976). Hybridization of RNA to double-stranded DNA: formation of R-loops. Proc Natl Acad Sci U S A, 73(7), 2294-2298.
Tippin, B., Pham, P., & Goodman, M. F. (2004). Error-prone replication for better or worse. Trends Microbiol, 12(6), 288-295.
Tse-Dinh, Y. C. (2015). Targeting bacterial topoisomerase I to meet the challenge of finding new antibiotics. Future Med Chem, 7(4), 459-471.
Tupper, A. E., Owen-Hughes, T. A., Ussery, D. W., Santos, D. S., Ferguson, D. J., Sidebotham, J. M., . . . Higgins, C. F. (1994). The chromatin-associated protein H-NS alters DNA topology in vitro. Embo j, 13(1), 258-268.
Usongo, V., & Drolet, M. (2014). Roles of type 1A topoisomerases in genome maintenance in Escherichia coli. PLoS Genet, 10(8), e1004543.
van de Putte, P., Westenbroek, C., & Rörsch, A. (1963). The relationship between gene-controlled radiation resistance and filament formation in Escherichia coli. Biochimica et Biophysica Acta (BBA) - Specialized Section on Nucleic Acids and Related Subjects, 76, 247-256.
Ventola, C. L. (2015). The Antibiotic Resistance Crisis: Part 1: Causes and Threats. Pharmacy and Therapeutics, 40(4), 277-283.
Voelkel-Meiman, K., Keil, R. L., & Roeder, G. S. (1987). Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I. Cell, 48(6), 1071-1079.
Vollmer, A. C., Belkin, S., Smulski, D. R., Van Dyk, T. K., & LaRossa, R. A. (1997). Detection of DNA damage by use of Escherichia coli carrying recA''::lux, uvrA''::lux, or alkA''::lux reporter plasmids. Appl Environ Microbiol, 63(7), 2566-2571.
Wada, M., Kano, Y., Ogawa, T., Okazaki, T., & Imamoto, F. (1988). Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli. J Mol Biol, 204(3), 581-591.
Wahba, L., Amon, J. D., Koshland, D., & Vuica-Ross, M. (2011). RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Mol Cell, 44(6), 978-988.
Walker, G. C. (1985). Inducible DNA repair systems. Annu Rev Biochem, 54, 425-457.
Wang, J. C. (2002). Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol, 3(6), 430-440.
Wehrli, W. (1983). Rifampin: mechanisms of action and resistance. Rev Infect Dis, 5 Suppl 3, S407-411.
Weinstein-Fischer, D., & Altuvia, S. (2007). Differential regulation of Escherichia coli topoisomerase I by Fis. Mol Microbiol, 63(4), 1131-1144.
Wen, C. Y. (2009). Regulation and Biological Functions of R-loop: Roles of RecF pathway and DNA topoisomerases. National Taiwan University.
Westover, K. D., Bushnell, D. A., & Kornberg, R. D. (2004). Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science, 303(5660), 1014-1016.
Wimberly, H., Shee, C., Thornton, P. C., Sivaramakrishnan, P., Rosenberg, S. M., & Hastings, P. J. (2013). R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli. Nat Commun, 4, 2115.
Xu, B., & Clayton, D. A. (1996). RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers. Embo j, 15(12), 3135-3143.
Yang, J., Annamalai, T., Cheng, B., Banda, S., Tyagi, R., & Tse-Dinh, Y. C. (2015). Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation. Antimicrob Agents Chemother, 59(10), 6195-6202.
Yim, G., McClure, J., Surette, M. G., & Davies, J. E. (2011). Modulation of Salmonella gene expression by subinhibitory concentrations of quinolones. J Antibiot (Tokyo), 64(1), 73-78.
Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E., & Lieber, M. R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol, 4(5), 442-451.
Zechiedrich, E. L., Khodursky, A. B., Bachellier, S., Schneider, R., Chen, D., Lilley, D. M., & Cozzarelli, N. R. (2000). Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli. J Biol Chem, 275(11), 8103-8113.
Zhang, Z. Z., Pannunzio, N. R., Hsieh, C. L., Yu, K., & Lieber, M. R. (2015). Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids. BMC Res Notes, 8, 127.