The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.308
ISSN1460-2075
AutoresHasna Boubakri, Anne Langlois de Septenville, Enrique Viguera, Bénédicte Michel,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle22 October 2009Open Access The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo Hasna Boubakri Hasna Boubakri CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France Université Paris-Sud, Orsay, FrancePresent address: Institut de Génétique et de Microbiologie, Université Paris-Sud, Orsay, F-91405, France. Search for more papers by this author Anne Langlois de Septenville Anne Langlois de Septenville CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France Université Paris-Sud, Orsay, France Search for more papers by this author Enrique Viguera Enrique Viguera Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain Search for more papers by this author Bénédicte Michel Corresponding Author Bénédicte Michel CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France Université Paris-Sud, Orsay, France Search for more papers by this author Hasna Boubakri Hasna Boubakri CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France Université Paris-Sud, Orsay, FrancePresent address: Institut de Génétique et de Microbiologie, Université Paris-Sud, Orsay, F-91405, France. Search for more papers by this author Anne Langlois de Septenville Anne Langlois de Septenville CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France Université Paris-Sud, Orsay, France Search for more papers by this author Enrique Viguera Enrique Viguera Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain Search for more papers by this author Bénédicte Michel Corresponding Author Bénédicte Michel CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France Université Paris-Sud, Orsay, France Search for more papers by this author Author Information Hasna Boubakri1,2, Anne Langlois de Septenville1,2, Enrique Viguera3 and Bénédicte Michel 1,2 1CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette, France 2Université Paris-Sud, Orsay, France 3Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain *Corresponding author. CNRS, Centre de Génétique Moléculaire, FRE 3144, UPR2167, 1 avenue de la Terrasse, Gif-sur-Yvette 91198, France. Tel.: +33 1 6982 3229; Fax: +33 1 6982 3140; E-mail: benedicte.mi[email protected] The EMBO Journal (2010)29:145-157https://doi.org/10.1038/emboj.2009.308 Correction(s) for this article The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo06 January 2010 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info How living cells deal with head-on collisions of the replication and transcription complexes has been debated for a long time. Even in the widely studied model bacteria Escherichia coli, the enzymes that take care of such collisions are still unknown. We report here that in vivo, the DinG, Rep and UvrD helicases are essential for efficient replication across highly transcribed regions. We show that when rRNA operons (rrn) are inverted to face replication, the viability of the dinG mutant is affected and over-expression of RNase H rescues the growth defect, showing that DinG acts in vivo to remove R-loops. In addition, DinG, Rep and UvrD exert a common function, which requires the presence of two of these three helicases. After replication blockage by an inverted rrn, Rep in conjunction with DinG or UvrD removes RNA polymerase, a task that is fulfilled in its absence by the SOS-induced DinG and UvrD helicases. Finally, Rep and UvrD also act at inverted sequences other than rrn, and promote replication through highly transcribed regions in wild-type E. coli. Introduction Replication fork arrest is a recognized source of genetic instability in all types of living cells. To limit the danger of replication arrest, eukaryotes induce checkpoint proteins that stabilize and protect blocked replication forks (reviewed in Branzei and Foiani, 2007; Tourriere and Pasero, 2007). Prokaryotes behave differently, they constitutively express replication restart proteins that are associated with the replication machinery (Sandler, 2000; Lecointe et al, 2007). Replication arrest can occur for many reasons, including collisions with DNA-bound proteins and particularly transcription complexes. As bacterial chromosomes are simultaneously transcribed and replicated, and because the velocity of the replication machinery (800 NT/s) is more than 10 times higher than that of the transcription machinery (50 NT/s), the problem raised by collisions between replication and transcription has been studied for decades (French, 1992; reviewed in Mirkin and Mirkin, 2007; Wang et al, 2007). Several in vitro and in vivo studies, showing that co-directional collisions do not seriously impede replication progression, lead to the conclusion that the replication machinery is not inactivated when it encounters an RNA polymerase transcribing the leading strand template (Pomerantz and O'Donnell, 2008 and references therein). In contrast, it is well established that head-on collisions between replication and transcription, that is the presence of an active RNA polymerase on the lagging strand template, arrest replication forks in vitro and in vivo (Deshpande and Newlon, 1996; Takeuchi et al, 2003; Mirkin and Mirkin, 2005). Genetic instability following head-on collisions of replication and transcription has been documented in bacteria and yeast (Vilette et al, 1995; Torres et al, 2004; Prado and Aguilera, 2005). To limit head-on collisions between replication forks and the highly expressed rRNA genes, yeasts and eukaryotic cells use replication fork barriers, which are DNA sites where binding of a specific protein prevents replication from entering the rDNA region in the direction opposed to transcription (Brewer et al, 1992). In bacteria, to avoid head-on collisions ribosomal operons (rrn) are transcribed in the direction of replication (Brewer, 1988; Rocha and Danchin, 2003). rrn operons are highly expressed and their promoter regions carry regulatory elements that adapt their level of expression to the growth rate, so that transcription is more efficient in rich than in minimal medium (MM) (Condon et al, 1992; reviewed in Paul et al, 2004). Ribosomal-RNA transcripts are not translated and premature transcription arrest is prevented by the association of the RNA polymerase with ‘an anti-termination’ machinery, which increases the transcription speed to 90 NT/s (reviewed in Condon et al, 1995). The universality of the presence of rrn operons on the leading strand template in bacteria suggests that rrn inversion impairs bacterial growth. Surprisingly, Escherichia coli viability was not affected by inverting large chromosomal regions that carry several rrn operons, even when the main homologous recombination DNA repair protein, RecA, was inactivated (Esnault et al, 2007). This observation suggested that bacteria encode proteins other than RecA that facilitates the progression of replication forks through oppositely oriented highly transcribed genes. We describe here the identification of helicases that have such a role. Helicases are enzymes that associate NTP hydrolysis with the capacity to translocate on DNA. Most helicases translocate on single-strand DNA (ssDNA) to unwind double-stranded DNA, several also unwind DNA–RNA hybrids, or can remove proteins from DNA during translocation. The first helicase described to remove an RNA polymerase from the path of replication forks was the T4 dda helicase (Bedinger et al, 1983). In yeast, this function is fulfilled by the superfamily 1 (SF1) helicase Rrm3, a 5′–3′ helicase required for efficient replication at numerous protein-bound sequences such as in rRNA and tRNA genes, centromeric and telomeric regions (Azvolinsky et al, 2006; reviewed in Boule and Zakian, 2006). In this study, we show that in bacteria the three helicases DinG, Rep and UvrD facilitate replication of the chromosome through oppositely oriented highly transcribed ribosomal operons. DinG belongs to the SF2 family of helicases and translocates in the 5′–3′ direction on ssDNA (Voloshin et al, 2003). In vitro, it unwinds a wide variety of substrates with a preference for D-loops and R-loops (Voloshin and Camerini-Otero, 2007). DinG is present in most prokaryotes and is related to the DNA helicases Chl1 and Rad3 from Saccharomyces cerevisiae, Rad15 from Schizosaccharomyces pombe and the human helicases XPD and BACH1 (Koonin, 1993; Rudolf et al, 2006; Voloshin and Camerini-Otero, 2007; Liu et al, 2008). Rad3 and XPD are components of the transcription factor IIH, they function in transcription initiation and nucleotide excision repair, and XPD defects are responsible for several human diseases (Liu et al, 2008 and references therein). Although DinG is an SOS-inducible protein (Lewis et al, 1992; Courcelle et al, 2001), its absence does not render E. coli sensitive to DNA damaging agents and to date the function of DinG in vivo is totally unknown. Rep and UvrD are also the founders of a large family of helicases, homologous to Srs2 in yeast. They belong to the SF1 superfamily, share 40% identity and translocate in the 3′–5′ direction on ssDNA. The uvrD gene was originally identified for its crucial role in nucleotide excision repair and mismatch repair. In addition, UvrD (but not Rep) can remove the replication terminator Tus protein from its cognate site, Ter, and the recombination protein RecA from ssDNA (Flores et al, 2005; Veaute et al, 2005; Bidnenko et al, 2006). Rep assists replication because in its absence chromosome replication takes twice as long when compared with wild-type cells, and arrested replication forks undergo a remodelling reaction called replication fork reversal (Lane and Denhardt, 1975; Seigneur et al, 1998). Rep was hypothesized to facilitate replication across DNA-bound proteins because it can dislodge a DNA-bound repressor during translocation in vitro (Yancey-Wrona and Matson, 1992). The rep uvrD double mutant is lethal and rescued by the inactivation of the pre-synaptic recombination proteins RecQ, RecJ and RecFOR (Petit and Ehrlich, 2002; Lestini and Michel, 2008); one of the physiological roles of UvrD is thus to remove RecQJFOR-dependent RecA filaments from stalled replication forks, or to prevent their formation. Finally, replication forks that have been inactivated restart with the use of the major restart protein PriA; in a priA mutant, replication restart is catalysed by an Rep-PriC-dependent pathway (Sandler, 2000; Heller and Marians, 2005). In this study, we show that chromosomal inversion, including E. coli rRNA operon(s) renders DinG essential for growth in rich medium. Moreover, the inactivation of the helicases DinG, Rep and UvrD has synergistic effects on replication blockage at an inverted rRNA locus. In the natural chromosome configuration, E. coli cells lacking these three helicases are viable only if the stability of the RNA polymerase is compromised and RecA binding is prevented by an RecF mutation. These results suggest that these helicases exert a fork-clearing function at inverted rrn loci and also at other transcription units. Results dinG inactivation confers rich medium sensitivity to strains that carry inverted rrn operons The lambda attR and attL attachment sites were used to construct strains carrying a chromosome inversion (Valens et al, 2004; Esnault et al, 2007). The InvA mutant carries a 18 kb inversion encompassing the rrnA operon (Figure 1). It carries only 11 genes in addition to rrnA, among which 4 are naturally oriented in opposition to replication. rrnA is the only transcription unit that is highly expressed and whose expression is increased in rich medium in InvA strains (Corbin et al, 2003; Lopez-Campistrous et al, 2005). The InvBE mutant carries a 138.3 kb inversion containing rrnB and rrnE; about 100 genes are present in the inverted region, among which 67% are originally co-directional with replication and may be sites of transcription–replication collisions after inversion. As rrn expression is growth-rate regulated, these two Inv mutants allowed the analysis of three kinds of head-on replication–transcription collisions: (i) in highly expressed rrn, (ii) in moderately expressed rrn, (iii) in genes other than rrn. As previously observed for similar inversions (Esnault et al, 2007), InvA and InvBE were fully viable on MM and on rich medium (Luria broth, LB) (Figure 2A and B; Supplementary Table S2). DinG, Rep and UvrD were inactivated in InvA and InvBE mutants to test whether these helicases are required for replication across oppositely oriented genes. Figure 1.Schematic representation of the inverted region in the mutants InvA (top) and InvBE (bottom). Numbers indicate the sequence coordinates in the wild-type E. coli MG1655 chromosome. The large black arrows indicate the inversion end points (lambda att sites). The grey arrows indicate the position of rrn operons (the coordinates of rrnA, and of rrnE and rrnB 3′ ends are indicated). The vertical arrows show the position of NotI sites (used for PFGE). Download figure Download PowerPoint Figure 2.The helicases Rep, UvrD and DinG are required for colony formation in Inv mutants. Appropriate dilutions of overnight cultures at 37°C in MM (OD 1.0–1.5) were plated on MM and LB plates, which were incubated at 37°C. Unmarked positions on the left of (A) (InvA), (B) (InvBE) and (C) (InvABE) are data points for Inv mutants that express all helicases. White boxes: colony forming units (cfu)/ml on MM plates after 48 h incubation; dark grey boxes: cfu/ml on LB plates after 16–24 h incubation; light grey boxes: cfu/ml on LB plates after 48 h of incubation. The hatched box indicates cfu/ml on MM after 3 days incubation. The results are also presented in Supplementary Table S2. Download figure Download PowerPoint dinG inactivation did not affect the formation of Inv mutant colonies on MM; however, colony formation on LB was strongly impaired for InvA dinG and delayed for InvBE dinG (Figure 2A and B). To test whether transcription is responsible for the LB sensitivity of InvA dinG and InvBE dinG mutants, we used the rpoCΔ215−220 mutation (called rpoC* thereafter). By mimicking the presence of ppGpp, this mutation reduces the stability of transcription elongation complexes (Bartlett et al, 1998, 2000; Trautinger and Lloyd, 2002; Trautinger et al, 2005). rpoC* restored 100% overnight colony formation on LB in both Inv dinG mutants (Figure 2C and D). In InvBE, the oriC-distal att site removes rrnB P1 Fis-binding sites (Supplementary Figure S1A), so that the promoter is weakened about seven-fold during steady-state growth in rich medium, but remains growth-rate regulated (Appleman et al, 1998; Hirvonen et al, 2001). Specifically, deleting the highly expressed rrnE operon in the InvBE dinG mutant fully restored the plating efficiency on LB (Supplementary Table S2). We conclude that DinG is required for efficient colony formation on rich medium when a highly expressed rrn operon is inverted on the chromosome, and that the growth defect observed in Inv dinG mutants is completely overcome by reducing the transcription level (growth on MM or inversion of only rrnB, which is deprived of Fis sites in this construction). rep inactivation causes cell elongation in rich medium Most of the rep mutants were constructed in the presence of a conditional Rep+ plasmid (IPTG dependent) that was cured before each experiment, (Supplementary Table S1; Lestini and Michel, 2008). Inactivation of rep in InvA or InvBE mutants did not cause any loss of plating efficiency (Figure 2A and B), although InvBE rep overnight colonies on LB were quite small. The introduction of rpoC* or the deletion of rrnE in InvBE rep suppressed this slow-growth phenotype, again suggesting a deleterious effect of the inverted highly expressed rrn operons (not shown). This idea was confirmed by the use of a strain with a large inverted region carrying the three operons rrnA, rrnB and rrnE (InvABE, 277.3 kb inverted): InvABE rep was sensitive to LB and this defect was fully suppressed by the rpoC* mutation (Figure 2C). In contrast, all Inv uvrD mutants were fully viable on LB as on MM (Figure 2). We conclude that the Rep helicase (and not UvrD) is required for colony formation on LB when at least three highly transcribed rrn operons are oriented opposite to replication. In contrast, a 277 kb inversion does not impair growth of the rep mutant providing that the rrn operons are only moderately expressed (InvABE cells grown in MM) or that the stability of the RNA polymerase is compromised (rpoC* mutant on LB, Figure 2). Formation of a visible colony requires about 24 generations and to determine whether the rep mutation affects Inv cell growth at early times, Inv rep cells were analysed by differential interference contrast (DIC) microscopy. Both InvA rep and InvBE rep cultures, shifted for 1 h from MM to LB, contained a high percentage of elongated cells, higher than rep or Inv single mutants (Table I). Cell elongation was weaker in MM and was strongly decreased by the rpoC* mutation or the deletion of rrnE and rrnB (from 29 to 3% in InvA rep and from 48 to 5–11% in InvBE rep, Table I), indicating that it is caused by the strong expression of inverted rrn. It was also specific for the rep mutation, as Inv uvrD cells were no more elongated than single uvrD mutants (6–11% elongated cells, Table I), and Inv dinG cells were not (InvBE) or only slightly (InvA) elongated (4 and 16% of elongated cells, respectively, Table I). The contrast between the elongated phenotype of Inv rep cells after a shift to LB and a wild-type efficiency of colony formation overnight on LB plates suggest an early defect followed by a recovery. Conversely, the absence of cell elongation of the Inv dinG mutants after a shift to LB contrasts with their plating defect suggests late, possible cumulative defects. These ideas were tested by analysing micro-colony formation by time-lapse microscopy (Supplementary Figure S2). InvA rep micro-colonies grown for a few hours on LB contained normal-sized cells, owing to the splitting of some elongated cells. Conversely, InvA dinG normal-sized cells growing on an LB agar pad produced micro-colonies composed of non-dividing, mostly elongated cells (Supplementary Figure S2). Table 1. Cell elongation after a shift to LB Strain Relevant genotype MMa LB 1 hb dinG rep uvrD 1 Nc 2 N >3 N 1 N 2 N >3 N JJC3524 + + + 73 27 (20) 0 62 (1) 36 (20) 2 InvA strains JJC4010/4802 + + + 58 41 (19) 1 (1) 61 36 (19) 3(2) JJC4678/4881S dinG + + 60 36 (12) 4 (1) 40 44 (12) 16 (8) JJC4408 + rep + 51 39 (8) 10 (9) 23 44 (5) 29 (12) JJC4873 + + uvrD 68 (1) 29 (1) 3 (3) 42 47 (16) 11 (7) JJC4880 dinG + uvrD 48 39 (1) 12 (2) 17 26 (2) 56 (7) JJC4828S dinG rep + 60 29 (3) 10 (6) 24 30 (1) 46 (3) JJC4879Sc + rep uvrD 11 25 64 (5) 4 24 72 (1) InvA rpoC* JJC4962 dinG + + 75 (5) 23 (13) 0 JJC4995 + rep + 64 (3) 33 (23) 3 (2) JJC4963 dinG + uvrD 54 (5) 30 (12) 17 (10) JJC4914S/4919 dinG rep + 54 (5) 33 (9) 13 (1) JJC5140S/5143 + rep uvrD 43 39 (5) 18 (0) InvA recA JJC4027 + + + 86 (7) 11 (3) 3 (1) JJC5040 + + uvrD 55 (2) 38 (15) 8 (3) JJC5042 dinG + uvrD 31 (1) 52 (9) 17 (1) JJC5053S + rep + 71 (0) 24 (5) 5 (4) 15 36 (2) 49 (5) InvA lexA JJC5096 + rep + 56 38 (12) 6 (4) 17 55 (4) 28 (9) InvBE strains JJC4349 + + + 83 (4) 17 (11) 1 (1) 52 (1) 40 (11) 7 (5) JJC4920 dinG + + 77 (1) 23 (14) 0 50 (1) 45 (13) 4 (2) JJC4700S/4978S + rep + 33 50 (9) 17 (13) 17 35 (3) 48 (10) JJC4870/4997 + + uvrD 78 (4) 22 (14) 0 45 48 (11) 7 (3) JJC4981 dinG + uvrD 63 (1) 34 (14) 3 (2) 25 43 (7) 27 (10) JJC4746S/5009S dinG rep + 52 34 (3) 14 (6) 7 37 (1) 56 (7) InvBE rpoC* JJC4987 + rep + 46 (1) 49 (23) 5 (3) JJC4966/4979 dinG + uvrD 55 (2) 26 (10) 18 (9) JJC4975 dinG rep + 58 (6) 36 (19) 5 (3) InvBE ΔrrnE JJC4951 dinG + + 80 (4) 19 (11) 0 JJC4973 dinG + uvrD 39 (0) 48 (11) 13 (5) InvBE ΔrrnE ΔrrnB JJC5125 dinG + + 82 (5) 19 (11) 0 JJC5154 + rep + 44 45 (18) 11 (10) JJC5158 dinG + uvrD 39 50 (11) 11 (10) JJC5156S dinG rep + 56 41 (14) 3 (3) JJC5157Sc + rep uvrD 80 (7) 19 (17) 1 30 35 (4) 34 (10) InvBE recA JJC4631 + + + 69 (7) 26 (15) 4 (1) JJC5036 dinG + + 59 (1) 36 (13) 4 (2) JJC5058S + rep + 35 43 (10) 22 (8) 24 36 (2) 40 (5) JJC5034 + + uvrD 54 (2) 30 (5) 15 (7) Non-inverted strains JJC3424 + + + 71 27 (20) 0 60 (1) 36 (20) 2 JJC4400 dinG + + 62 36 (12) 1 (1) JJC4984 + rep + 35 49 (8) 16 (13) JJC4858 + + uvrD 74 25 (16) 1 42 52 (19) 6 (3) JJC4872 dinG + uvrD 45 45 (1) 10 (5) JJC4804S dinG rep + 83 (6) 6 (4) 4 (2) JJC4878Sc + rep uvrD 45 44 11 (3) 25 39 (2) 36 (3) Non-inverted rpoC* JJC5164S/5165 + rep uvrD 63 (1) 26 (15) 11 (3) JJC4629 recA 79 (6) 16 (5) 2 (1) ‘S’: the pAM-rep plasmid was cured before the experiment, the strain number is followed by an ‘S’ to indicate that experiment was performed after the plasmid has been segregated. In each medium, the smallest wild-type cells produced by division (baby cells, 1.5 μM in MM and 2.1 μM in LB) were used as cell unit and their size was, as expected, half that of the smallest cells with a detectable septum. Numbers indicate the percentage of cells in each of the following categories: 1 N: cells whose length was from baby wild-type cells to twice as long; 2 N: cells whose length was between twice and three times that of baby wild-type cells; >3 N: cells longer than three times the size of baby wild-type cells. With few exceptions, 150–300 cells were counted. Numbers in parentheses indicate the percentage of cells with a visible septum in formation. Data in bold differ at least three-fold from their parental values (InvA and InvBE single mutants, and non-inverted cells carrying the same helicase or recA mutations); for these mutants, results are the average of two independent experiments. a Cells in exponential phase in MM. b Cells in exponential phase shifted for 1 h in LB. c JJC4879, JJC5157 and JJC4878 are rep uvrD recF mutants. The effects of the dinG, rep and uvrD mutations are additive To analyse whether DinG, Rep and UvrD have independent or overlapping roles, we tested whether the inactivation of two of these three helicases is synergistic. Cells that do not carry a chromosome inversion were tested first, showing that dinG uvrD and dinG rep double mutants are fully viable (Figure 2E). As described earlier (Petit and Ehrlich, 2002), non-inverted rep uvrD cells were (i) nearly lethal on MM (small colonies appeared in 3 days), (ii) lethal on LB and (iii) mainly rescued by recF inactivation (Figure 2E). Therefore, Inv rep uvrD mutants were tested in a recF mutant background. As recF inactivation per se does not affect the growth of Inv strains (Supplementary Table S3, see below) and is beneficial to rep uvrD cells, we consider thereafter that the growth defects of rep uvrD recF mutants carrying an inversion result from the inactivation of the rep and uvrD genes and not from the recF mutation. All Inv mutants lacking two helicases were sensitive to rich medium as they formed colonies on LB plates with a very low efficiency (Figure 2A and B). As the InvA dinG mutant was already quite sensitive to rich medium, the deleterious effect of inactivating uvrD in this mutant can be deduced from the increased level of elongated cells after only 1 h of propagation in LB (Table I). A high percentage of elongated cells are observed in all Inv mutants lacking two helicases. It is accompanied by a decrease in the number of cells with a visible septum (number in parenthesis in Table I), in agreement with a cell division defect. Therefore, dinG, rep and uvrD mutations are synergistic, indicating overlapping functions. Inv dinG uvrD mutants were fully viable on MM whereas a significant plating defect of both Inv dinG rep mutants on MM indicates replication impairment by moderately expressed rrn in this mutant and suggests overlapping functions of Rep and DinG (MM, Figure 2A and B). Inv rep uvrD recF cells were also impaired on MM; the plating defect was stronger for InvBE than for InvA, suggesting a possible replication impairment also at non-rrn sequences (MM, Figure 2A and B; Inv rep uvrD RecF+ colonies were not obtained). rrn expression is responsible for the growth defects of helicase mutants on LB rpoC* and rrn deletion alleles (ΔrrnE and ΔrrnB, Supplementary Figure S1) were used to ascertain the role of rrn in the observed growth defects. rpoC* was first tested in a non-inverted rep uvrD mutant. Importantly, rpoC* rescued colony formation of rep uvrD cells on MM and on LB, regardless of the recF status (Figure 2E). This result indicates that (i) in E. coli the presence of both Rep and UvrD is required because of a high level of transcription and (ii) decreasing transcription by affecting the stability of RNA polymerase bypasses the need for RecFOR inactivation. In InvA mutants, rpoC* restored the viability of both dinG uvrD and dinG rep cells (although InvA dinG uvrD rpoC* remained slightly impaired on LB) and the InvA rep uvrD rpoC* mutant formed colonies on LB in 2 days (Figure 2C–E; Table I). Therefore, the growth defects of all the three InvA mutants lacking two helicases result from the high level of rrnA expression. In InvBE dinG rep, introduction of the rpoC* allele improved viability and decreased cell elongation in LB, as observed for the InvA strain (Figure 2D; Table I). Accordingly, deletion of both rrnE and rrnB also fully rescued the InvBE dinG rep mutant, confirming that these highly expressed operons are the only deleterious sequences in this mutant (Figure 2D, Table I). In contrast, rpoC* did not rescue InvBE dinG uvrD, but deletion of both rrnE and rrnB allowed a full recovery of colony formation on LB (Figure 2D; Table I; Supplementary Table S2). These observations allow us to conclude that rrn are also the only deleterious sequences in InvBE dinG uvrD, but that even in the presence of the rpoC* mutation, inverted rrn impair growth of this mutant on rich medium. In InvBE rep uvrD cells, introduction of rpoC*, deletion of rrnE or of both rrn allowed colony formation on MM but cells remained sensitive to LB, even in a recF context (Figure 2D; Supplementary Table S2). Therefore, the inversion of genes other than rrn is deleterious in rich medium in rep uvrD and rep uvrD recF mutants. The requirement for UvrD in Inv dinG mutants is not because of its anti-RecF-RecA action An recF null mutation was used to test whether UvrD is required in Inv dinG mutants to counteract a deleterious DNA binding of RecFOR, and in turn RecA. recF inactivation did not improve the growth of dinG, uvrD or dinG uvrD Inv mutants (Supplementary Table S3). We conclude that in Inv dinG uvrD mutants, the deleterious effect of the absence of UvrD is not because of the lethal binding of RecFOR-RecA to DNA. We propose that the synergistic effects of dinG and uvrD inactivation in cells carrying a highly expressed inverted rrn operon reflect a redundant function of these two helicases. In agreement with a previous report, we observed that recA inactivation did not affect the viability of InvA and InvBE single mutants (Esnault et al, 2007; Supplementary Table S3). However, recA deletion prevented growth of InvBE dinG and Inv rep mutants on LB (Supplementary Table S3). Furthermore, no plasmid-less colony could be obtained from Inv dinG rep recA; [pAM-rep] cells even on MM, indicating that in both Inv backgrounds the dinG rep recA combination of mutations is lethal (Supplementary Table S3; uvrD recA colonies were slow growing on LB and were not affected by inversion, Supplementary Table S3). This suggests that the lack of Rep and/or DinG in Inv mutants generates ssDNA that renders homologous recombination and/or SOS induction crucial for viability. Notably, in Inv rep mutants the inactivation of the SOS response by a lexAind mutation also delayed (InvA) or prevented (InvBE) colony formation on LB (Supplementary Table S3; Supplementary Figure S2), indicating that the plating defect of Inv rep recA mutants may mainly result from the absence of SOS induction. The combination of rep uvrD dinG recF mutations is lethal in non-inverted strains and rescued by rpoC* We attempted to construct a rep uvrD dinG recF mutant by eliminating the pAM-Rep+ plasmid from rep uvrD dinG recF [pAM-Rep+] cells. Small plasmid-less colonies were obtained in 3 days on MM but some failed to grow in overnight cultures and others exhibited variable plating efficiencies, indicating that the simultaneous inactivation of the three helicases Rep, UvrD and DinG is nearly lethal in a recF E. coli mutant (Supplementary Table S2). Therefore, the viability of each helicase double mutant relies on the presence of the third helicase when all genes are in their original orientation. The rpoC* mutation also failed to restore rep uvrD dinG co
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