Zeynep ; Baharoglu

Roxane Lestini

Stéphane Duigou

Bénédicte

Bénédicte Michel
email: benedicte.michel@cgm.cnrs-gif.fr

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases

Keywords: replication restart, RNA polymerase, helicase, stringent response, replication restart, RNA polymerase, helicase, stringent response

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases

INTRODUCTION

Replication forks are susceptible to be arrested by a variety of obstacles, including DNA-bound proteins such as RNA polymerases (RNA Pol) [START_REF] Mirkin | Replication fork stalling at natural impediments[END_REF][START_REF] Rudolph | Avoiding and resolving conflicts between DNA replication and transcription[END_REF]. DNA instability is associated with replication fork arrest (reviewed in [START_REF] Aguilera | Genome instability: a mechanistic view of its causes and consequences[END_REF], and in order to limit the deleterious consequences of replication-transcription collisions, cells encode enzymes that facilitate replication through transcription units. In yeast, the Rrm3 helicase travels with the replication fork machinery and dislodges RNA Pols from tRNA and rRNA genes as well as tightly bound proteins from heterochromatin (Azvolinsky et al., 2009 and references therein). In E. coli, this function was originally ascribed to the Rep helicase; firstly, based on the observations that in the rep mutant chromosome replication is slowed down and requires specific functions [START_REF] Lane | The rep mutation. IV. Slower movement of replication forks in Escherichia coli rep strains[END_REF][START_REF] Petit | Essential bacterial helicases that counteract the toxicity of recombination proteins[END_REF][START_REF] Seigneur | RuvAB acts at arrested replication forks[END_REF], and secondly, because the purified Rep helicase is specifically capable of dislodging DNA-bound proteins [START_REF] Yancey-Wrona | Bound Lac repressor protein differentially inhibits the unwinding reactions catalyzed by DNA helicases[END_REF]. More recently, evidence was provided that transcribed sequences are indeed a major obstacle to replication in rapidly growing cells, and that Rep is the main, though not the only helicase that assists replication progression across highly transcribed sequences [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF].

Rep and UvrD are two 3' to 5' helicases that share 40% homology. The existence of a redundant function for these paralogues has been suspected since the original observation that the rep uvrD double mutant shows severe growth defects [START_REF] Taucher-Scholtz | Functions of helicases in E. coli. In Mechanisms of DNA replication and recombination[END_REF].

However, the situation turned out to be more complex and UvrD is now known to play two different roles in rep mutants, revealed by the two classes of mutations that suppress the growth defects of the rep uvrD double mutant. The first class of mutations that was identified inactivates the RecFOR recombination pathway [START_REF] Petit | Essential bacterial helicases that counteract the toxicity of recombination proteins[END_REF]. Combined with the observation that UvrD can remove RecA from ssDNA in vitro, the rescue of rep uvrD cells by the inactivation of recombination proteins led to the proposal that UvrD is essential in rep mutants because it removes deleterious RecFOR-dependent RecA-filaments that assemble at blocked replication forks [START_REF] Lestini | UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli[END_REF][START_REF] Veaute | UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli[END_REF]. However, the nature of the obstacles arresting replication in the first place, thus allowing RecFOR-RecA binding to forks in rep mutants, remained unknown. Moreover, the suppression of rep uvrD colethality by recFOR inactivation was shown to be only partial, confirming the existence of an obstacle to replication restart other than RecFOR-RecA bound to DNA in rep uvrD cells [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF]. Two lines of evidence identified RNA polymerases as the original cause of replication arrest in rep uvrD cells: first, mutations that map in the RNA Pol genes rpoB and rpoC were shown to suppress the growth defects of this double mutant [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF], second, replication arrest sites were directly visualized in rep and rep uvrD recF mutants at inverted ribosomal operons (rrn), provided that these operons were facing replication and were highly transcribed [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF].

Rep was confirmed to be the major accessory helicase and was shown to be attracted to replication forks by a direct interaction with the replicative helicase DnaB [START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF].

In addition to UvrD, a third player was identified, the 5' to 3' helicase DinG [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. DinG is essential for the viability of the rep uvrD recF mutant and it was proposed that, provided that recF is inactivated, DinG can clear RNA Pols from blocked replication forks in the absence of both Rep and UvrD. In addition, DinG is essential for the viability of rep and uvrD single mutants when replication collides with RNA Pol at highly-expressed inverted rrn, indicating that the presence of two of these three helicases is required when replication-transcription collisions are increased. Finally, DinG has the specific function of unwinding RNA-DNA hybrids in vivo, since it is also essential for viability when replication forks are arrested by R-loops [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. The described RNA Pol mutations that suppress rep uvrD growth defects are mutations that constitutively confer phenotypes akin to the induction of the stringent response [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF]. The stringent response is an adaptation to amino acid starvation through the induction of the alarmone ppGpp (reviewed in [START_REF] Potrykus | p)ppGpp: still magical?[END_REF]. Binding of ppGpp to RNA Pol, as well as mutations that mimic this binding, affect transcription initiation from specific promoters, which decreases the expression of ribosomal operons (rrn) and activates the expression of amino acids biosynthetic genes in relA spoT double mutants, allowing their growth in minimal medium without amino acids (Barker et al., 2001a;Barker et al., 2001b;[START_REF] Bartlett | RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters[END_REF][START_REF] Bartlett | Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters[END_REF][START_REF] Zhou | The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli[END_REF].

The so-called "stringent mutations" in RNA Pol mimic the presence of ppGpp in decreasing the half-life of open complexes. Although we have not measured the half-life of transcription open complexes, we will call here "stringent-like" the phenotype conferred by RNA Pol mutations that both decrease rrn expression in rich medium and allow growth of relA spoT double mutants on minimal medium. RNA Pol mutations that increase transcription of amino acid biosynthetic promoters also destabilize transcription elongation complexes (TEC) [START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF][START_REF] Trautinger | RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription[END_REF]. Such mutations were thought to suppress the lethality of rep uvrD double mutants owing to the destabilisation of RNA Pol-DNA complexes [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF].

In this work, we report that rep uvrD recF cells grow poorly on rich medium at low temperature and we isolated five mutations suppressing this LB-cryo-sensitivity. Similarly to the mutations that suppress the growth defects of rep uvrD mutants at 37°C, the suppressor mutations isolated here in rep uvrD recF mutants at 30°C map in rpoB and rpoC. One of these mutations is close to the active site; the others are in, or close to, the primary DNA-RNA binding channel, suggesting that they affect the stability of transcription complexes on DNA. Only one of these mutations exhibits a stringent-like phenotype, showing that our assay

RESULTS

rep uvrD recF mutants are cryo-sensitive

We constructed rep uvrD recF triple mutants in the presence of a conditional plasmid that carries the rep wild-type gene (pAM-rep; [START_REF] Lestini | UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli[END_REF]. This plasmid only replicates in the presence of the Lac promoter inducer, isopropyl thio-galactoside (IPTG) and is cured upon cell propagation in the absence of IPTG. We analyzed the properties of rep uvrD recF cells cured of pAM-rep. For historical reasons the work was realized part in an AB1157 background (classically used for homologous recombination studies) and part in an MG1655 background (the more generally used, sequenced wild-type strain). In the AB1157 background, colony formation was delayed on LB at 37°C and 30°C; at 25°C only a variable subpopulation of cells formed colonies (JJC4048, Table I). In the MG1655 background, results were similar except for a partial defect of plating efficiency on LB at 30°C (JJC5136 and JJC5166 Table I). Finally, we constructed an Hfr strain which allowed the co-introduction of the three rep uvrD recF mutations by conjugation, and observed that in this Hfr-PK3-PO131 background the growth defect was more pronounced than in other backgrounds, as plasmid-less rep uvrD recF cells were not recovered with the expected efficiency even on minimal medium (MM) at 37°C (not shown). In order to understand the reasons for the cryosensitivity of rep uvrD recF mutants, we studied five AB1157 rep uvrD recF suppressed clones which are able to form large colonies on LB at 30°C in 2 days. The rpoC Δ215-220 mutation, previously shown to restore the viability of rep uvrD cells at 37°C (Boubakri et al., Analysis of the subpopulation of Hfr plasmid-less colonies formed in two days at 37°C on MM supplemented with casamino acids revealed that one of them was resistant to rifampicin. Rif R mutations map in rpoB, the gene coding for the β subunit of RNA Pol, which suggested that this particular rep uvrD recF clone carries an rpoB mutation that suppresses the rep uvrD recF growth defect (JJC4100, Table S1). To ascertain that the Rif R mutation was responsible for the improved growth of the rep uvrD recF Rif R clone, pAM-rep was reintroduced and the suppressed clone was P1 transduced with thiC::Tn10, a locus close to the rpoB rpoC genes. As expected, the Rif resistance phenotype was 90% linked with thiC::Tn10 (43/48 Tet R transductants were Rif S ). Curing of pAM-rep showed that rep uvrD recF thiC::Tn10 clones that had remained Rif R had kept the capacity to form colonies overnight (ON) at 37°C on LB whereas the thiC::Tn10 clones that had lost the Rif R phenotype grew as poorly as the original Hfr rep uvrD recF mutant. This mutation is therefore necessary and sufficient for the improved viability of rep uvrD recF cells. Sequencing of the rpoB gene revealed the presence of a mutation, N518D, in the rpoB Rif R cluster 1.

Overnight cultures of AB1157 rep uvrD recF cells were plated on LB at 30°C. Five colonies isolated in three independent experiments were kept for further studies (called S1, S2, and S3a, S3b, S3m). Replacement in the five suppressed strains of the rep uvrD recF region of AB1157 by that of the Hfr JJC4100, or of the rep::Ap R allele by a rep::cm R allele, did not modify their growth properties (Table S1, data not shown). To determine whether the isolated mutations map in the rpoBC genes, a Tet S derivative of each suppressed clones was A plasmid carrying an IPTG-inducible rpoB + gene was introduced in Ap S derivatives of the suppressed clones and viability was measured in the presence of IPTG (Table S2).

Expression of RpoB rendered S3m cryo-sensitive on LB, suggesting that this mutant carries a recessive rpoB mutation; sequencing rpoB showed the presence of a H447R mutation. In S3a, the control vector pUC19 could not be introduced whereas the plasmid pUC-rpoB + transformed with a normal efficiency (not shown); nevertheless, transformants remained cryoresistant (Table S2). Sequencing rpoB revealed the presence of a D444G mutation. These observations suggest that, in addition to suppressing the rep uvrD recF growth defect, the rpoB D444G mutation somehow prevents pUC propagation. This defect in plasmid propagation is recessive (as it is complemented in cis by the wild-type rpoB + allele) whereas the suppression of rep uvrD recF cryo-sensitivity is dominant over the wild-type allele. In S1 and S3b expression of rpoB + did not prevent growth at 25°C (Table S2), so we sequenced rpoC in these two mutants and found a P451L mutation (S1), and a H113R mutation (S3b). Finally, both rpoB and rpoC genes were sequenced in S2. rpoB was intact and rpoC carried the H113R mutation, which was thus obtained twice independently. To our knowledge, the three rpoB D444G , rpoC H113R and rpoC P451L mutations have not been described previously, whereas the rpoB N518D allele has been already isolated in a screen for Rif R clones [START_REF] Garibyan | Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome[END_REF], and the rpoB H447R allele in a screen for mutations that increase the expression of amino acid biosynthetic genes in a relA spoT context [START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF].

The rpo sup mutations are necessary and sufficient for the suppressor phenotype

To determine whether the rpo mutations isolated here (called rpo sup thereafter) are necessary and sufficient for the suppressor phenotype, these mutations were transferred to a rep uvrD recO strain by P1 co-transduction with thiC::Tn10 [RecF is known to act in conjunction with two other proteins, RecO and RecR [START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF], and inactivation of either the RecF, RecO or RecR protein allows colony formation of rep uvrD cells at 37°C on MM with casamino acids [START_REF] Petit | Essential bacterial helicases that counteract the toxicity of recombination proteins[END_REF]]. rep uvrD recO cells harboring the plasmid pBGts-rep were used for strain construction; this plasmid carries the wild-type rep gene and can be cured by growing cells at 42°C [START_REF] Petit | Essential bacterial helicases that counteract the toxicity of recombination proteins[END_REF]. The phenotype of plasmid-less cells obtained after propagation at 42°C was analyzed. The five rep uvrD recO rpo sup mutants formed about 100% colonies on LB at 37°C, 30°C and 25°C, as the original rep uvrD recF rpo sup cells (Table 1). We conclude that the rpo sup mutations are necessary and sufficient to restore full viability to rep uvrD recF (recO) cells at low temperatures.

RecQ acts in concert with RecFOR to promote RecA binding to blocked forks in the rep uvrD mutant [START_REF] Lestini | UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli[END_REF]. We observed that the rep uvrD recQ mutant is more sensitive to LB than rep uvrD recF (recO) cells (Table I), suggesting that the RecFOR proteins still bind to arrested forks in a rep uvrD recQ mutant. The rpo sup alleles suppressed the LB-sensitivity of rep uvrD recQ cells, with the notable exception of the Rif R (rpoB N518D ) mutation that suppressed only at 37°C (Table 1). The residual cryo-LB-sensitivity of the rep uvrD recQ rpoB N518D indicates that RecFOR bind blocked replication forks in the absence of RecQ in this particular RNA Pol mutant, and that this mutation is a less efficient suppressor than the others at low temperature, in agreement with its original isolation at 37°C. Nevertheless, all other rpo sup mutations and the rpoC Δ215-220 The rpoC Δ215-220 mutation alters the kinetic properties of transcription complexes, reducing rRNA transcription and increasing transcription from some amino acid biosynthetic genes [START_REF] Bartlett | RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters[END_REF]. On the other hand, it behaves as most of the mutations isolated here since it suppresses the growth defects of rep uvrD recF, recO and recQ cells at all temperatures [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF] (Table 1). In addition, one of the mutations isolated here (rpoB H447R ) was also previously isolated in a screen for mutations that confer a stringent-like phenotype [START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF]. These observations prompted us to test whether the rpo sup mutations isolated here prevent the stimulation of the rrnB core promoter after a shift to rich medium. The rpo sup mutations were co-transduced with thiC::Tn10 into a strain carrying a P1 rrnB -lacZ fusion, which was used to compare the expression of the P1 rrnB core promoter in MM and in LB [START_REF] Bartlett | RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters[END_REF]. As expected, in wild-type cells the expression of lacZ from P1 rrnB promoter was higher in LB than in MM, whereas P1 rrnB activity remained low in both media in the presence of the rpoC Δ215-220 or rpoB H447R mutations (Fig. 1). This experiment revealed that rpoB D444G also reduces rRNA expression in LB (Fig. 1). In contrast, P1 rrnB expression remained higher in LB than in minimal medium in the presence of the rpoB N518D , rpoC P451L , and rpoC H113R alleles as in wild-type cells, showing that none of these three mutations affects the activity of rRNA promoter.

RelA and SpoT proteins are implicated in ppGpp alarmone synthesis. relA spoT double mutants do not induce the stringent response and are thus unable to grow on MM unless amino acids are provided. RNA Pol mutations that mimic the stringent response are classically isolated as suppressing the auxotrophy of relA spoT mutants. Although this has not been actually demonstrated, it was suggested that the inability of relA spoT mutants to grow on MM and the ability of the rpoBC suppressor mutations to suppress this defect results from effects of the mutations on transcription of amino acid biosynthetic operons [START_REF] Paul | DksA potentiates direct activation of amino acid promoters by ppGpp[END_REF] see Rutherford et al., 2009, for discussion). In order to analyse the capacity of the rpo sup ΔrelA::Kan R mutants (strains JJC 4553 to JJC4559 Table S1) and P1-transduced them with a ΔspoT::Cm R null mutation, plating half of the transduction mixture on LB and half on MM.

All rpo sup ΔrelA::Kan R mutants could be transduced by the ΔspoT::Cm R null mutation on LB while only one mutant, rpoB H447R ΔrelA::Kan R , provided transductants on MM (Table S3). As expected from this result, none of the rpo sup ΔrelA::Kan R ΔspoT::Cm R mutants obtained on LB could grow on MM except for the rpoB H447R ΔrelA::Kan R ΔspoT::Cm R strain (Table S3).

The growth defect on MM was specific for a relA spoT context, since none of the RNA Pol mutations prevented growth on MM in RelA + SpoT + cells (see plating efficiencies on MM in Fig. 3 and Fig. 4). This result shows that only the rpoB H447R mutation prevents the increase of rrn expression in rich medium and allows growth of a relA spoT mutant on MM. The rpoB D444G mutation only affects rrn expression and the other rpo sup mutations exhibit none of these stringent-like phenotypes. This result shows that four of the five mutations isolated in this study would not be obtained in a classical screen for mutations that restore growth of a relA spoT mutant on MM. Although they do not confer a stringent-like phenotype, the rpo sup mutations bypass the need for accessory replication helicases, suggesting that they affect the stability of transcription complexes without affecting their kinetic properties on rrn and amino acid biosynthetic gene promoters. The possible instability of the mutant RNA Pol-DNA complexes was tested by two different genetic approaches.

rpo sup mutations improve the resistance to UV irradiation of a ruv mutant and the rpoC H113R mutation rescues a greA greB double mutant.

RuvABC is a recombination complex that acts at the last step of homologous recombination by resolving recombination intermediates called Holliday junctions [START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF]. ruv mutants are hyper-sensitive to UV irradiation and rpoB or rpoC mutations that exhibit a stringent-like phenotype partially relieve this hyper-sensitivity. It was proposed that they increase the intrinsic instability of RNA Pol-DNA complexes when RNA Pol is blocked by a DNA lesion [START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF]. A ruvABC deletion was introduced in all rpo sup mutants and the UV resistance of the resulting strains was measured.

With the exception of the rpoC H113R allele, which was poorly viable in a ruv mutant context and yielded variable results, all mutations improved the UV resistance of the ruvABC mutant (Fig. 2A). This result supports the idea that the mutations isolated here affect the stability of RNA Pol-DNA complexes, either at promoters or in transcription elongation complexes (TEC).

When RNA Pol encounters a block during elongation and backtracks, the transcription factors GreA and GreB suppress pausing by stimulating the intrinsic nucleolytic activity of RNA Pol (reviewed in [START_REF] Borukhov | Bacterial transcription elongation factors: new insights into molecular mechanism of action[END_REF]. greA greB double mutants are non-viable at high temperature, presumably because prolonged RNA polymerase pausing prevents replication and/or transcription. A mutation isolated through its stringent-like phenotype was previously reported to suppress the thermo-sensitivity of greA greB mutants [START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF]. greA greB rpo sup mutants were constructed and tested for growth at 42°C. Only the rpoC H113R mutation allowed the growth of greA greB mutant at 42°C (Fig. 2B). This result presumably reflects the destabilisation of backtracked RNA Pol by the rpoC H113R , but this level or type of destabilisation is not essential for the growth of rep uvrD recF cells at 30°C since it is observed for only one of the suppressor mutations. This result also indicates that mutations that do not affect rrn or amino acid biosynthetic gene expression can nevertheless decrease the stability of stalled TEC enough to rescue a greA greB mutant. Altogether, we infer from the phenotypes conferred by the RNA Pol mutations described here that these mutations compromise the stability of transcription complexes; the consequences of this

Several rpo sup mutants rescue rep uvrD mutants in a RecF + context

If the viability of rep uvrD recF rpo sup mutants results from less replication arrest, or from facilitated replication restart, the inactivation of recF might not be needed for viability.

Actually, certain RNA Pol mutations including the rpoC Δ215-220 allele were reported to suppress the growth defect of rep uvrD cells in a RecF + context at 37°C [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF]. To construct rep uvrD rpo sup mutants, rep and uvrD were introduced in rpo sup single mutants containing pGBts-rep, and/or the rpo sup mutation was co-transduced with thiC::Tn10 to a rep uvrD [pAM-rep] mutant. The viability of plasmid-less clones was measured after curing cells by propagation at 42°C (pGBts-rep) or in the absence of IPTG (pAM-rep) (Fig. 3A). The rpoC H113R mutation allowed formation or rep uvrD colonies on LB at all temperatures. The rpoC P451L mutant yielded slow-growing colonies that were heterogeneous in size at low temperature. Two of the mutations that affect rrn expression (rpoC Δ215-220 , rpoB H447R ) restored rep uvrD cells viability on LB, but colony formation was delayed at low temperature and also slightly decreased for rpoC Δ215-220 at 25°C. In the rpoB D444G context, colony formation on LB was delayed at all temperatures and significantly decreased at 25°C. Finally, the rpoB N518D mutation had only a partial effect at 37°C and did not allow colony formation at low temperatures. These observations indicate that in one rep uvrD rpo sup mutant (rpoC H113R ) RecFOR does not bind replication forks at any temperature. In the other rep uvrD rpo sup mutants, although RecFOR is not lethal any more as in rep uvrD Rpo + cells, it remains partly deleterious, slowing down and/or preventing growth, mainly at low temperature. Therefore, the modifications of RNA Pol activity caused by the different mutations determine the accessibility of replication forks to RecFOR recombination proteins.

mutations rescue rep uvrD recF dinG mutants but not rep uvrD dinG mutants

The viability of the rep uvrD recF mutant relies on the presence of a third helicase called DinG [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. We previously reported that the rpoC Δ215-220 mutation suppresses the lethality of rep uvrD recF dinG mutants at 37°C on MM and on LB, but not that of rep uvrD dinG mutants, indicating that it facilitates replication restart in the absence of all three accessory helicases only if RecF does not poison arrested forks [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. We tested whether the rpo sup mutations also suppress rep uvrD recF dinG lethality by constructing rep uvrD recF dinG rpo sup mutants in two ways, first a dinG deletion was introduced in the original rep uvrD recF rpo sup clones (JJC4043 to JJC4047, context AB1157, Table S1), and secondly the rpo sup mutations were introduced in a rep uvrD recF dinG mutant (JJC5405 to JJC5426, context MG1655, Table S1). Results were similar in both backgrounds (data not shown and Fig. 3B). rpoC P451L was the only mutation that did not suppress the lethality of rep uvrD recF dinG cells as no plasmid-less cells could be obtained (not shown).

The best suppressor mutation was again the rpoC H113R which restored 100% rep uvrD dinG recF plating efficiency at 37°C (although colony formation was delayed) and at low temperature (Fig. 3B). The three mutations rpoC Δ215-220 , rpoB H447R and rpoB D444G also restored 100% plating efficiency but colony formation was delayed on LB at 37°C for rpoB D444G , on LB at all temperatures for rpoC Δ215-220 , and in all growth conditions for rpoB H447R . Finally, rpoB N518D allowed normal colony formation on MM but formed only about 10% of heterogeneous slow-growing colonies on LB at 37°C and did not suppress the rep uvrD recF dinG lethality on LB at low temperature.

It was previously reported that rpoC Δ215-220 only rescues a rep uvrD dinG mutant that lacks RecF [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. To address this question for the rpo sup mutants, rep uvrD dinG rpo sup RecF + mutants were constructed, firstly by introducing the dinG deletion in the S1) and secondly by introducing successively all three helicase deletions in the rpo sup mutants (JJC4911 to JJC4918, JJC5310 and JJC5311, context AB1157, Table S1). All strains yielded similar results: the rpo sup mutations did not suppress the lethality of rep uvrD dinG mutants in a RecF + context, as either no plasmid-less colonies could be recovered after growth under non-permissive conditions for plasmid replication (rpoB N518D ), or only a few plasmid-less colonies were obtained which could not be propagated and eventually acquired additional suppressive mutations, possibly because the uvrD context is mutator (data not shown). In conclusion, in the absence of all three helicases the rpo sup mutations restore cell viability only if recF is inactivated, as previously observed for the rpoC Δ215-220 mutation in a MG1655 background at 37°C [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. This result indicates that, at least in the absence of the three Rep, UvrD and DinG helicases, replication forks are still arrested by the encounter of the rpo sup mutated RNA polymerases, allowing RecFOR to gain access to DNA. This result also indicates that DinG is responsible of the viability of rep uvrD rpo sup DinG + mutants shown in Fig. 3A, since the inactivation of dinG in this context is lethal.

To further test the effects of the rpo sup mutations on replication-transcription collisions, we introduced these mutations in cells where such collisions are increased by a chromosome rearrangement.

Rescue of helicase mutants that carry an inverted rrn operon depends on the rpo mutation.

Inversion of an rrn operon creates a region of increased head-on collisions between replication and transcription. Such inversions render the dinG mutant sensitive to rich medium because of R-loop formation, and strongly impair growth of the rep dinG double mutant, even on MM, because of DNA Pol-RNA Pol collisions [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF] (Fig. ). In a strain that carries an inverted rrnA operon (InvA) the rpoC Δ215-220 mutation suppresses the growth defects of both dinG and dinG rep mutants at 37°C, on MM and on LB, presumably by decreasing the transcription efficiency of rrnA [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF] (Fig. 4A).

We tested whether the rpo sup mutations decrease the formation of R-loops in inverted rrn by introducing these mutations in InvA dinG cells and measuring plating efficiencies of the resulting combination of mutations on LB. rpoC Δ215-220 , previously tested at 37°C only, was tested here at 30°C. The rpoC H113R mutation and the three mutations that affect rrn expression (rpoB H447R rpoB D444G and rpoC Δ215-220 ) restored full viability of the InvA dinG mutant at 37°C and 30°C (Fig. 4A). Therefore, they decrease R-loop formation in this context at both temperatures. In contrast the Rif R mutation (rpoB N518D ) allowed InvA dinG colony formation on LB at 37°C but not at 30°C, and the rpoC P451L mutations had no effect. The phenotype of this last mutant suggests that suppression of the rep uvrD recF LB-cryosensitivity by rpo sup mutations is independent of R-loop removal. To ascertain directly that the LB-cryo-sensitivity of the rep uvrD recF mutant does not result from R-loop formation, a plasmid that over-expresses RnaseH (pEM001) was introduced in this mutant. As shown in Table 1, this plasmid did not improve plating efficiency. In conclusion, most of the rpo sup mutations suppress or decrease R-loop formation in InvA cells, allowing InvA dinG viability, but conversely suppression of R-loops is neither necessary nor sufficient to improve growth of rep uvrD recF cells at 30°C on LB.

rpo sup mutations were also introduced in an InvA dinG rep mutant, where replication is arrested in the inverted rrnA by collisions with RNA Pol [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. As previously reported the mutation rpoC Δ215-220 restored a normal plating efficiency on MM and LB [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and as expected the rpoB D444G mutation, which decreases Prrn efficiency (Fig. 1), exhibited a similar phenotype (Fig. 4B). Surprisingly, the rpoB H447R mutation improved colony formation only on MM. It is conceivable that the decrease of rrn expression measured in Fig. 1 with a promoter deprived of its FIS binding sites is compensated by the presence of these sites at inverted rrnA [START_REF] Bartlett | Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters[END_REF]. Finally, rpoC H113R also improved viability on MM whereas the rpoC P451L and rpoB N518D mutations had no effect. Altogether, these results indicate that in rich medium only two mutations, rpoC Δ215- 220 and rpoB D444G destabilize RNA Pol enough to allow replication across an inverted rrn in the absence of Rep and DinG, i.e. when only the UvrD helicase is active. Among the suppressor mutations that do not affect rrn expression, rpoC H113R is the only one that rescues an InvA dinG rep mutant, and only on MM.

A consequence of replication arrest in the rep mutant is a reaction called replication fork reversal, in which the two ends of the newly synthesised strands at a blocked replication fork anneal to form a DNA double-strand end and a Holliday junction. This reaction renders RecBC (the recombination enzyme specific for DNA double-strand ends) essential for viability [START_REF] Michel | Recombination proteins and rescue of arrested replication forks[END_REF][START_REF] Seigneur | RuvAB acts at arrested replication forks[END_REF]. Interestingly, although this study and other recent studies suggest that in the rep mutant replication forks are arrested by transcribed sequences, none of the mutations isolated here rescued the viability of rep recB cells, as no plasmid-less colonies could be recovered after propagation of rep recB rpo sup [pAM-rep] mutants in the absence of IPTG (data not shown, strains JJC5532 to JJC5536 in Table S1).

This indicates that the rpoB mutation does not prevent replication fork reversal in a rep mutant, and that, even when the RNA Pol is mutated and weakly bound to DNA, its removal from DNA by UvrD and/or DinG takes place within the context of restarting reverted replication forks. We show here that transcription is a stronger obstacle to replication at lower temperatures than at 37°C and we isolated mutations that suppress the rich medium cryosensitivity of rep uvrD recF cells. The genetic properties of these mutants suggest that the rep uvrD and rep uvrD recF strains provide a new way of isolating mutations that decrease the stability of RNA Pol on DNA, at promoters and/or in TEC. Furthermore, the analysis of these RNA Pol mutants reinforces the notion that Rep, UvrD and DinG have redundant functions in E. coli to facilitate the progression of replication forks across transcribed sequences.

DISCUSSION

Isolation and properties of new RNA Pol alleles.

The atomic structure of Thermus thermophilus and Termus aquaticus RNA Pol have led to structural models of transcription elongation complexes (TEC) [START_REF] Korzheva | A structural model of transcription elongation[END_REF][START_REF] Vassylyev | Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution[END_REF]Vassylyev et al., 2007a;Vassylyev et al., 2007b;[START_REF] Zhang | Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution[END_REF].

The similarity of these RNA Pols with the E. coli enzyme allows us to map the residues affected in our rpoB and rpoC mutants on these structures. The position of the mutated amino acids on a representation of a wild-type transcription complex is shown in Fig. 5. The three rpoB mutations, D444G, H447R and N518D (D324, H327 and T398 in T. thermophilus and T. aquaticus) lie in the major channel for the DNA/RNA hybrid, in, or close to the β-lobe 1, in a region previously described to be important for the stability of RNA Pol-DNA complexes [START_REF] Rutherford | Allosteric control of Escherichia coli rRNA promoter complexes by DksA[END_REF][START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF]. The rpoC H113R mutation (H90 in T. thermophilus and H101 in T. aquaticus) is on the other side of the major channel and although the mutated residue is buried in the enzyme, it may affect interactions of RpoC with the hybrid DNA-RNA. rpoC P451L (P719 in T. thermophilus and P730 in T. aquaticus) is in a different location in the complex, in a highly conserved region around the active site. This mutation is adjacent to a mutation isolated as a ΔdksA suppressor (H450R) and, since the RNA Pol cofactor DksA is also involved in the stability of transcription complexes, mutations in this region could affect the enzyme in another way to those which flank the major channel [START_REF] Rutherford | Allosteric control of Escherichia coli rRNA promoter complexes by DksA[END_REF]. In spite of the proximity of this mutation to the active site, we did not detect any deleterious effect of the rpoC P451L allele on E. coli growth (data not shown).

Biochemical analysis of these mutant enzymes would tell whether the in vivo instability of transcription complexes is related to an increased propensity to pause and/or to terminate transcription.

The phenotypes of the isolated RNA Pol mutations do not allow their classification in specific groups but allow us to rank them according to the advantage that they confer to cells that lack Rep, UvrD and/or DinG helicases (summarised in Table 2). The best suppressor of growth defects in the absence of these helicases is the rpoC H113R mutation, which was isolated twice independently. This RNA Pol mutant is the only one that forms TEC unstable enough to suppress the temperature sensitivity of a greA greB double mutant. It confers LB resistance to rep uvrD, rep uvrD dinG recF and InvA dinG cells at all temperatures, and improves growth of InvA dinG rep cells at 37°C on MM. Of all mutants isolated here, the RpoC H113R RNA Pol is probably the one that forms the weakest complex on DNA. It is also the only one that slows down the growth of otherwise wild-type cells (data not shown), and could not be reliably combined with a ruvABC deletion. Slightly less efficient than rpoC H113R , the mutations rpoB H447R and rpoB D444G confer an intermediate phenotype, as the previously described rpoC Δ215-220 allele. The destabilisation of transcription complexes caused by these mutations, which is deduced from their stringent-like phenotype and from the increased UV resistance conferred to ruv mutants, allows rep uvrD dinG recF and rep uvrD cells to grow on LB. They rescue InvA dinG and InvA rep dinG mutants, with the exception of the LB sensitivity of InvA rep dinG rpoB H447R cells (discussed below). Finally, the two less efficient suppressors are rpoC P451L and rpoB N518D . rpoC P451L is the only mutation that is close to the active site, while rpoB N518D was isolated at 37°C. The only sign that these mutated RNA Pols might be weakly bound to DNA, beside the rescue of rep uvrD recF cells, is the increase of UV Several mutations that mimic the effects of ppGpp have been isolated by different means, most often because they activate amino acid biosynthetic genes in a relA spoT double mutant, enabling it to grow on minimal medium [START_REF] Bartlett | RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters[END_REF][START_REF] Broccoli | Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation[END_REF][START_REF] Szalewska-Palasz | Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54[END_REF][START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF][START_REF] Zhou | The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli[END_REF]. Some of these mutations decrease expression from the rrn promoter regardless of the presence of the upstream FIS binding sites [START_REF] Szalewska-Palasz | Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54[END_REF], while the FIS sites compensate for the rpoC Δ215-220 mutation effects on the rrnB promoter [START_REF] Bartlett | Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters[END_REF]. One mutation conferring a stringent-like phenotype was shown to reduce R-loop formation in a nontranslated sequence by causing premature transcription arrest in vivo [START_REF] Broccoli | Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation[END_REF], and two others were shown to decrease the stability of TEC in vitro [START_REF] Trautinger | RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription[END_REF].

Therefore, the rpoC Δ215-220 , rpoB D444G and rpoB H447R mutations may be affected for the stability of transcription initiation complexes, or for the stability of TEC, or for both. Because the FIS sites compensate for the rpoC Δ215-220 mutation effects on the rrnB promoter, this mutation is likely to rescue helicase mutants carrying an inverted rrn by destabilizing TEC, as proposed in [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. rpoB D444G may rescue InvA dinG rep cells because the promoter remains weakly expressed on LB in the presence of the FIS Sites, or because TEC are unstable. The RpoB H447R RNA Pol may fail to rescue InvA dinG rep cells because its defects are compensated for by the presence of the FIS sites and it forms more stable TEC than the RpoC Δ215-220 RNA Pol during rrn transcription. This observation supports the idea that there is no necessary correlation between the instability of TEC and the instability of the open complexes on rrn promoters. Furthermore, we isolated here a mutant (rpoB D444G ) that affects transcription initiation at rrn promoters without affecting the expression of amino acids biosynthesis genes in a relA spoT context. To our knowledge, such a mutant was not reported before. Finally, rescue of a greA greB mutant at high temperature was previously described for a RNA Pol mutation isolated as increasing amino acids biosynthetic genes expression [START_REF] Trautinger | Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase[END_REF] whereas here it is observed for a RNA Pol mutant that does not affect these genes, nor rrn expression (rpoC H113R ).

We used strains carrying a chromosome inversion to measure the consequences of a weaker stability of transcription complexes when the rate of replication arrest is increased either by the formation of R-loops, or by encountering oppositely-oriented highly-active RNA Pol in inverted rrn. With the exception of rpoC P451L , all RNA Pol mutations isolated here prevent R-loop formation. This means that RNA Pols that form unstable transcription complexes are less prone than the wild-type enzyme to R-loop formation. Conversely, only one (rpoB D444G ) could prevent the lethality associated with the encounter of a series of RNA Pol transcribing a ribosomal operon in the direction opposite to replication. This means that even RNA Pols weakly bound to DNA will arrest replication forks when the latter collide with an oppositely-oriented highly-transcribed operon.

How does E. coli deal with replication-transcription collisions?

The general scheme that emerges from the present study and from previous studies of rep, dinG and uvrD single and multiple mutants is that Rep is the first factor that facilitates replication through transcribed sequences in vivo [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF][START_REF] Lane | The rep mutation. IV. Slower movement of replication forks in Escherichia coli rep strains[END_REF][START_REF] Petit | Essential bacterial helicases that counteract the toxicity of recombination proteins[END_REF][START_REF] Seigneur | RuvAB acts at arrested replication forks[END_REF]. In the absence of Rep, UvrD becomes essential for E. coli viability and mutations in RNA Pol suppress this colethality, which points to UvrD as the main back up to Rep for RNA Pol removal [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF][START_REF] Guy | Rep provides a second motor at the replisome to promote duplication of protein-bound DNA[END_REF]this work). Two lines of evidence point to DinG as the other helicase that acts as a back up. The first, is our previous report that even in the absence of RecF, the combination of the rep, uvrD, and dinG mutations is lethal in E. coli expressing the wild-type RNA Pol [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF]. This suggests that DinG removes wild-type RNA Pols from replication forks when Rep, UvrD and RecF are absent. The second, is our present observation that DinG is required for suppression of the rep uvrD co-lethality by most of the rpo sup mutations studied here, indicating that because they are unstable on DNA, the mutant RNA Pol can be removed from replication forks by DinG regardless of the presence of RecF.

However, these mutated RNA Pols do not suppress the need for RecBC in a rep mutant, indicating that DinG and UvrD act in the context of restarting reversed replication forks.

These helicases may recognize some features of reversed or of the restarting replication forks that are not shared by the original replication fork, whereas Rep may act within the context of the original replication fork that encounters the obstacle. It should be noted that the rpo sup mutations do not prevent spontaneous replication arrest in the E. coli chromosome since they did not relieve the growth defects of a priA mutant, which lacks the main replication restart protein PriA (strains JJC5540 and JJC5541 in Table S1, data not shown).

At low temperature, even in the presence of DinG, rep uvrD recF cells grow poorly on LB, indicating that DinG has a limited ability to remove the wild-type RNA Pol from replication forks under these growth conditions. Either transcription is a stronger obstacle to replication at low temperature, for example because transcriptional complexes are more stable, or for some reason DinG is less active. Provided that RecF is absent, several RNA Pol mutations isolated here bypass not only the need for Rep and UvrD but also the need for DinG (rep uvrD dinG recF rpo sup are viable, Fig. 3B). Removal of the mutant RNA Pol may then be catalyzed either by some yet unknown function or by the replisome itself. Actually in a purified system in vitro the wild-type RNA Pol initiation complex can be dislodged by the replisome [START_REF] Pomerantz | Direct restart of a replication fork stalled by a head-on RNA polymerase[END_REF]. However, since RecF remains lethal in a rep uvrD dinG rpo sup mutant, replication forks are still arrested in these rpo sup cells that lack the It should be noted that UvrD can act both ways, it removes RecA from DNA or prevents RecFOR binding, depending on the cause of replication arrest [START_REF] Lestini | UvrD controls the access of recombination proteins to blocked replication forks[END_REF]. The hypothetical removal of RecFOR and/or RecA from DNA by DinG needs to be tested in vitro; nevertheless, we do not favour this hypothesis because it is difficult to explain why DinG would remove RecF only in certain RNA Pol mutants. We favour the hypothesis that DinG and RecFOR compete for blocked replication forks in rep uvrD cells. Because DinG is unable to remove the wild-type RNA Pol, rep uvrD cells are killed by RecFOR. Similarly, the rep uvrD rpo N518D mutant is non-viable on LB at low temperature because under these conditions DinG cannot dislodge this mutated RNA Pol from DNA, letting RecFOR bind to arrested replication forks. Conversely, DinG is capable of removing all other RNA Pol mutants isolated here before RecFOR binds, and consequently the viability of the rep uvrD mutant becomes independent of the recF context in these RNA Pol mutants.

Experimental procedures

Strains and plasmids

The strain backgrounds are MG1655, or JJC40, which is an hsdR Thr + Pro + derivative of AB1157 (leu-6 thi-1, his-4, argE3, lacY1, galK2, ara-14, xyl-5, mtl-1, tsx-33, rpsL31, supE44, hsdR, hsdM). JJC147 is leuB6,tonA1,lacY1,supE44, S1. InvA is described in [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], it is an MG1655 derivative, deleted for lacZ and attλ, which carries a 18kb inversion (NT 4 025 300 to 4 043 723) encompassing the rrnA operon. Minimum medium is M9 supplied with thiamine 0.05%, CaCl 2 100 µM, MgSO 4 2 mM, glucose 0.4% [START_REF] Miller | A short course in bacterial genetic: Cold Spring Harbor[END_REF]. Casamino acids 0.2% were added for strain background other than MG1655.

Most of the strains were constructed by P1 transduction [START_REF] Miller | A short course in bacterial genetic: Cold Spring Harbor[END_REF]. For Hfr conjugations, a mix of 10% donor cells / 90% recipient cells was incubated for 15 min with low agitation at 37°C before plating on selective media. Null mutants were checked by PCR with external oligonucleotides that amplify a DNA fragment of different length for the wild-type and the interrupted alleles. Oligonucleotides used for checking mutations are shown in Table S4. uvrD mutants were checked for their UV sensitive phenotypes and for their mutator phenotype (about 100-fold increase in the proportion of Rif R clones in overnight cultures).

The plasmids pGBts-rep [START_REF] Petit | Essential bacterial helicases that counteract the toxicity of recombination proteins[END_REF], pAM-rep [START_REF] Lestini | UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli[END_REF] and pAM-priA [START_REF] Grompone | PriA is essential for viability of the Escherichia coli topoisomerase IV parE10(Ts) mutant[END_REF] were described; they carry the rep or the priA gene under the control of their own promoter, they were segregated as published.

rpo mutations are rpoB A1331G (rpoB D444G ), A1340G (rpoB H447R ), A1552G (rpoB N518D ), rpoCA338G (rpoC H113R ), C1352T (rpoC P451L ). Following P1 transduction or conjugation, screening for A1552G (rpoB N518D ) was based the Rif R phenotype it confers.

Screening for A1331G (rpoB D444G ) was done sequencing the rpoB region. Three mutations were screened after PCR amplification of the region either by sequencing or by looking for the presence of a restriction site that they create: A1340G (rpoB H447R ) creates a BsiEI site, rpoCA338G (rpoC H113R ) creates a BssHII site, C1352T (rpoC P451L ) creates a BspM1 site.

Upon P1 co-transduction of rpo sup with thiC::Tn10 in a context where the mutation confers a phenotype, the phenotype and the presence of the mutation were tested in four to eight different Tet R transductants and there was 100% correlation between the presence of the Measurement of plating efficiency. Overnight cultures grown in MM (OD 650 1 to 2) were diluted and plated on MM or LB plates, incubated at 37°C, 30°C or 25°. LB plates at 37°C were counted after 24 and 48 hours of incubation, MM plates at 37°C and LB plates at 30°C after 48 hours and 3 days of incubation, and MM plates at 30°C and LB plates at 25°C after 3 and 4 days incubation.

Measurement of UV sensitivity and of β-galactosidase activity.

Survival to UV irradiation was performed as described [START_REF] Baharoglu | RuvAB is essential for replication forks reversal in certain replication mutants[END_REF]. βgalactosidase assays for the measure of P rrnB activity were performed as described [START_REF] Miller | A short course in bacterial genetic: Cold Spring Harbor[END_REF]. were previously published [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and were reproduced here as a control.

Legends to Figures

Plating efficiencies on MM 37°C are not shown and were similar to those at 30°C. [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and were reproduced here as a control. Δ215-220, P451L and H113R are rpoC mutations, D444G, H447R and N518D are rpoB mutations (MG1655 context). Symbols are as in Fig. 3, dark blue are plating efficiencies on MM at 37°C. B -Three rpo sup mutations restores growth of InvA dinG rep cells at 37°C on MM, only rpoB D444G also restores viability on LB. Results for the InvA rep dinG rpoC Δ215-220 mutant were previously published [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and were reproduced here as a control.

-

In addition to the phenotypes indicated here, all mutations rescue a rep uvrD recF (recO) mutant on LB at all temperatures, all rescue a rep uvrD recQ mutant on LB (except rpoB N518D at low temperature), all improve the UV resistance of a ruvABC mutant (except rpoC H113R which could not be reliably combined with the ruvABC mutation) and none rescue the lethality of a rep uvrD dinG triple mutant. MM or on LB, at 37°C or 30°C). Mutants are in the MG1655 context, similar results were obtained in the AB1157 context (not shown). Results for the rep uvrD dinG recF rpoC∆215-220 mutant at 37°C were previously published [START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and were reproduced here as a control. Plating efficiencies on MM 37°C are not shown and were similar to those at 30°C. 80x129mm (300 x 300 DPI)

  way of isolating RNA Pol mutants that are weakly bound to DNA, in transcription initiation or elongation complexes. Furthermore, these RNA Pol mutants allow us to extend our study of helicases that assist replication progression across transcription obstacles.

  and a suppressor mutation identified in the Hfr rep uvrD recF context at 37°C were included in this study.Suppressors of rep uvrD recF LB-cryo-sensitivity map in rpoC and rpoB genes.

  thiC::Tn10. For each mutant 7 to 11 out of 12 thiC::Tn10 transductants became cryo-sensitive, indicating that these five suppressor mutations are linked to thiC.

  the auxotrophy of relA spoT double mutants, we constructed rpo sup

  further investigated in backgrounds that lack different accessory helicases.

  rep uvrD rpo sup mutants previously made RecF + (JJC5253 to JJC5257 context AB1157 and JJC5258 context JJC4100, Table

  ruv mutants. In their presence, rep uvrD cells form slow growing colonies only at 37°C, and InvA dinG rep cells remain sick on MM. Furthermore, rpoC P451L fails to rescue InvA dinG cells on LB, while rpoB N518D only rescues it at 37°C and does not rescue rep uvrD recQ cells at 30°C.

  which is another indication that the dislodging of RNA Pol takes place during replication restart. Our study of RNA Pol mutants that form unstable complexes on DNA provided conditions where viability is dependent on DinG only when RecF is present (the rep uvrD rpo sup combinations of mutations become LB sensitive upon DinG inactivation only in a RecF + context). This observation suggests that DinG counteracts a deleterious action of RecF; either DinG could remove RecFOR / RecA from DNA, or it could prevent RecFOR binding.

  Tn10). Strains genotypes and plasmids are described in Table

  appearance of the new phenotype. 20 regularly spaced oligonucleotides in rpoB (10 on each strand) and 20 regularly spaced oligonucleotides in rpoC (10 on each strand) were used for sequencing of the entire genes.

Figure 1 .

 1 Figure 1. Two of the five rpo sup RNA Pol mutations exhibit a stringent-like phenotype. βgalactosidase assays were performed on strains carrying a P 1rrnB -lacZ fusion and an rpo sup mutation. The height of the histograms indicates β-galactosidase Miller Units, vertical bars indicate standard deviations. Wt stands for wild-type, Δ215-220 is the control rpoC Δ215-220

Figure 2 .

 2 Figure 2.A -Four rpo sup RNA Pol mutations improve growth of UV irradiated ruvABC

Figure 3 .

 3 Figure 3. A. rpo sup RNA Pol mutations restore growth of rep uvrD cells. The height of the

Figure 4 .

 4 Figure 4. A -Three rpo sup RNA Pol mutations restore growth of InvA dinG cells at all

Figure 5 .

 5 Figure 5. Schematic representation of the RpoB and RpoC subunits of RNA Pol showing the position of the rpo sup mutations. In orange RpoB, in green RpoC. Blue and red lines represent

Figure 1 .

 1 Figure 1. Two of the five rposup RNA Pol mutations exhibit a stringent-like phenotype. βgalactosidase assays were performed on strains carrying a P1rrnB-lacZ fusion and an rposup mutation. The height of the histograms indicates β-galactosidase Miller Units, vertical bars indicate standard deviations. Wt stands for wild-type, ∆215-220 is the control rpoC∆215-220 mutation. P451L and H113R are rpoC mutations, D444G, H447R and N518D are rpoB mutations. Light grey: cells grown in MM; dark grey: cells grown in LB. 80x59mm (300 x 300 DPI)

Figure 5 .

 5 Figure 4. A -Three rposup RNA Pol mutations restore growth of InvA dinG cells at all temperatures.The height of the histograms indicates the number of cfu/ml, vertical bars indicate standard deviations. Results for the InvA dinG rpoC∆215-220 mutant at 37°C were previously published[START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and were reproduced here as a control. ∆215-220, P451L and H113R are rpoC mutations, D444G, H447R and N518D are rpoB mutations (MG1655 context). Symbols are as in Fig.3, dark blue are plating efficiencies on MM at 37°C. B -Three rposup mutations restores growth of InvA dinG rep cells at 37°C on MM, only rpoBD444G also restores viability on LB. Results for the InvA rep dinG rpoC∆215-220 mutant were previously published[START_REF] Boubakri | The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo[END_REF], and were reproduced here as a control. 80x140mm (300 x 300 DPI)

  mutation suppress the residual growth defects of rep uvrD recQ cells.

	Suppression of the LB-cryo-sensitivity of rep uvrD recF does not correlate with a stringent-
	like phenotype of these RNA Pol mutants.

Table 1 :

 1 rpoB sup and rpoC sup mutations suppress the LB-cryo-sensitivity of rep uvrD recF, rep uvrD recO and rep uvrD recQ.. Numbers in italics indicate the formation of small colonies appearing 24 hours later than wt. JJCn-S indicates that the strain JJCn was used after segregation of the Rep encoding plasmid. A fresh plasmid-less colony was used for each experiment and cured clones were not kept. a = context MG1655. All other strains are in an AB1157 context. pEM001 is a plasmid that over-expresses RNase H.

	37°C	30°C	25°C

Table 2 .

 2 Summary of the phenotypes conferred by the different rpoB/C mutations.

		P1rrnB	relA	greA	rep uvrD	rep uvrD	InvA dinG	InvA dinG
		in LB	spoT on	greB at	RecF + (LB)	dinG recF	(LB)	rep (LB)
			MM	42°C		(LB)
	rpoBC+	high	-	-	-	-	-	-
	rpoC H113R rpoC Δ215-220	high low	-+ (a)	+ -	+ + (∼cryoS) + (delayed) +	+ +	-(MM +) +
	rpoB D444G	low	-	-	delayed and cryoS	+	+	+
	rpoB H447R	low	+	-	+ (delayed	+ (delayed)	+	-(MM +)
					at low T°)
	rpoC P451L	high	-	-	+ (delayed)	-	-	-
	rpoB N518D	high	-	-	cryoS delayed and	cryoS	cryoS

Acknowledgements

We thank O. Espeli, R.L.G. Gourse and C.J. Herbert for very helpful reading of the manuscript. We are very grateful to Annie Kolb for discussions on RNA polymerase structure, and to Audrey Lagès and Marie Le Masson for excellent technical assistance. The work was supported by ANR-05-BLAN-0204-01, ANR-08-BLAN-0230-01 and Prix "coup d'élan" from the foundation Bettencourt-Schueller. RL is supported by the Fondation de la Recherche Médicale (FRM grant number FDT 20070910238), and ZB by a PhD grant from the French ministry of research.


Dr Atul Mehta
email: atul.mehta@royalfree.nhs.uk

Joe T R Clarke

Roberto Giugliani

Perry Elliott

Aleš Linhart

Michael Beck

Gere Sunder-Plassmann

P Rozenfeld

K Nicholls

J Kleinert

A-C Hauser

O Bodamer

W Kristoferitsch

W Schreiber

B Plecko

T Kroepfl

I P Kotanko

C Binder

M Brunner-Krainz

Graz ) Belgium

C Verellen-Dumoulin

F Dehout

D Roland

F Henry

J Jaumotte

F Eyskens

A Neirinck

M C Nassogne

Y Pirson

A Ballaut

P Goyens

M Libert

L Kalakun

C Netto

T Vieira

L Jardim

B Lemieux

D Bichet

M F Arthus

Montreal C Fortier

C Azcona

M West

K Lemoine

A Chan

Edmonton E Kowal- Zuk

Vancouver L Clarke

L Golánv

J Bultas

J-C Lubanda

S Magage

D Karetová

G Dostalova

G Choukroun

S Carey Reomonnay

Cambrai Benziane

Montpellier A Khau Van Kien

J M Mittelberger

Freyming Merlebach

C Thevenot

E Hachulla

D Dobbelaere

B Dussol

Maubeuge R Reade

P Kaminsky

C Guyot

M Lino

Nice T Ghafari

E Monlun

D P Germain

N Ouali

G Touati

O Lidove

B Knebelmann

R Perrichot

Saint Lô B Richalet

Selestat V Klotz

E Andres

K Baron

S Delgado-Sanchez

R Hartung

G Kalkum

C Whybra

C Kampmann

C Wiethoff

A Schwarting

A Keilmann

S Pitz

K Bruns

F Bähner

K Lackner

B Koletzko

B Hoffmann

Munich B Pontz

H P H Neumann

M Cybulla

G Schluh

Freiburg A Winter

A Das

S Illsinger

T Lücke

A Von Arnim-Baas

J Hennermann

N Weinhold

J-M Berger

Cottbus S Stolz

S Grabbe

T Jansen

E Chrobot

A Gal

E Schäfer

K Ullrich

N Muschol

I Berkau

A Rolfs

T Böttcher

S Miethe

F Rimmele

C Wanner

I Davydenko

R Wössner

S Al Sawaf

O Gabrielli

D Concolino

W Borsini

R Parini

S Sala

M Rigoldi

R Di

A P Burlina

Natural course of Fabry disease: changing pattern of causes of death in FOS -the Fabry Outcome Survey

Keywords: Agalsidase alfa, enzyme replacement therapy, Fabry disease, outcomes database, pharmacoepidemiological survey

Background

Fabry disease is a rare X-linked lysosomal storage disorder characterized by severe multisystemic involvement that leads to major organ failure and premature death in affected men and women. Over the past 7 years, the Fabry Outcome Survey (FOS) has collected data on the natural history of Fabry disease and the long-term efficacy and safety of enzyme replacement therapy. This paper provides an update since the first analysis of FOS data.

Design

Baseline data on clinical manifestations and causes of death in a cohort of 1453 patients (699 males) from 19 countries worldwide were analysed. Causes of death of affected relatives were analysed separately.

Results

The most frequently reported signs and symptoms of Fabry disease were neurological.

Cardiac, ocular, gastrointestinal, dermatological, auditory and renal manifestations were also common. The principal causes of death among 181 affected relatives of patients in FOS (the majority of whom had died before 2001) were renal failure in males (42%) and cerebrovascular disease in females (25%). In contrast, among the 42 patients enrolled in FOS whose deaths were reported between 2001 and 2007, cardiac disease was the main cause of death in both males (34%) and females (57%).

Conclusion

These data suggest that the importance of renal disease as a cause of death in patients with Fabry disease is decreasing, while the importance of cardiac disease is increasing. This pattern likely reflects improvements in the management of renal disease in patients with Fabry disease.

INTRODUCTION

Fabry disease is a rare X-linked lysosomal storage disorder due to deficient activity of the enzyme α-galactosidase A. It is characterized by severe multisystemic involvement that ultimately leads to major organ failure and premature death in affected men and women [START_REF] Desnick | The metabolic and molecular basis of inherited disease[END_REF]. The condition has conventionally been considered to be rare, affecting only 1 in 40,000-117,000 live male births [START_REF] Desnick | The metabolic and molecular basis of inherited disease[END_REF][START_REF] Meikle | Prevalence of lysosomal storage disorders[END_REF]. However, a recent screening study suggested a much higher incidence of 1 in 3100-4600 individuals [START_REF] Spada | High incidence of later-onset fabry disease revealed by newborn screening[END_REF]. α-Galactosidase A deficiency is caused by a number of mutations affecting the GLA gene, many of which are private (i.e. occurring in only single or small numbers of families) [START_REF] Schafer | Thirty-four novel mutations of the GLA gene in 121 patients with Fabry disease[END_REF]. As a result of α-galactosidase A deficiency, there is progressive accumulation of undegraded glycosphingolipids, predominantly globotriaosylceramide (Gb 3 ), within cell lysosomes throughout the body. Common clinical features of Fabry disease include acroparaesthesia and pain crises, gastrointestinal symptoms, angiokeratomas and corneal dystrophy.

Despite the fact that Fabry disease follows an X-linked pattern of inheritance, it is now widely accepted that female heterozygotes can be severely affected, although progression of the disease to organ failure generally occurs later in life and symptom severity tends to be milder and more variable than in males [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF][START_REF] Deegan | Natural history of Fabry disease in females in the Fabry Outcome Survey[END_REF][START_REF] Wilcox | Females with Fabry disease frequently have major organ involvement: lessons from the Fabry Registry[END_REF]. Renal failure has been found to be a frequent cause of death in several studies, particularly in males [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF][START_REF] Branton | Natural history of Fabry renal disease: influence of alpha-galactosidase A activity and genetic mutations on clinical course[END_REF][START_REF] Linhart | Cardiac manifestations of Anderson-Fabry disease: results from the international Fabry outcome survey[END_REF]. In females, data on small patient groups suggest that cardiac disease and cerebrovascular disease are the most frequent causes of death [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF][START_REF] Linhart | Cardiac manifestations of Anderson-Fabry disease: results from the international Fabry outcome survey[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF][START_REF] Whybra | A 4-year study of the efficacy and tolerability of enzyme replacement therapy with agalsidase alfa in 36 women with Fabry disease[END_REF]. Death occurs on average 15 years earlier in females and 20 years earlier in males compared with the general population [START_REF] Deegan | Natural history of Fabry disease in females in the Fabry Outcome Survey[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females[END_REF][START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males[END_REF].

Enzyme replacement therapy (ERT) has become an established treatment for Fabry disease following demonstrations of both efficacy and safety in adults and children [START_REF] Schiffmann | Enzyme replacement therapy in Fabry disease: a randomized controlled trial[END_REF][START_REF] Eng | Safety and efficacy of recombinant human alpha-galactosidase A replacement therapy in Fabry's disease[END_REF][START_REF] Ramaswami | Enzyme replacement therapy with agalsidase alfa in children with Fabry disease[END_REF][START_REF] Wraith | Safety and efficacy of enzyme replacement therapy with agalsidase beta: an international, open-label study in pediatric patients with Fabry disease[END_REF]. Treatment guidelines concerning ERT have now been published [START_REF] Eng | Fabry disease: guidelines for the evaluation and management of multi-organ system involvement[END_REF]. Early diagnosis of Fabry disease is therefore important, and clinicians in a range of specialties, as well as general practitioners, should be aware of the signs, symptoms and natural history of the disease.

This paper provides an update of the demographic and baseline clinical characteristics of a large cohort of patients with Fabry disease enrolled in FOS -the Fabry Outcome Survey -and examines the causes of death among patients and their affected relatives [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF]. using an adapted version of the Mainz Severity Scoring Index (FOS-MSSI) [START_REF] Whybra | Measurement of disease severity and progression in Fabry disease[END_REF].

METHODS

Patients and data collection

Data analysis

Student's two-sample and one-sample t-tests and Fisher's exact/Chi-square tests were used for statistical analyses, using SAS version 9.1 (Cary, NC). Values are presented as mean (SD) unless otherwise stated; p<0.05 was considered significant.

RESULTS

Patient demographics

Table 1 shows the baseline characteristics of the 1453 patients (699 males) enrolled in FOS at the time of this analysis (December 2007). The majority were Caucasian (95.7%).

Disease severity

The severity of Fabry disease was greater in males than in females in FOS, as indicated by the FOS-MSSI total score [16.9 (10.8) The 42 patients in FOS who died were severely affected by Fabry disease. From the available absolute data shown in Table 4, both men and women who died had higher MSSI scores than patients who were alive. Data were also available on whether patients had specific manifestations either before or at/after the start of ERT (information recorded as yes/no only). Among the 42 deceased patients, LVH was reported in 29 cases (25 male, 4 female). LVH was present before the start of treatment in all 22 of the patients who went on to receive ERT and in 7 of the 9 patients who never received ERT. Absolute data on left ventricular mass (LVM) were available for 19 of the 42 patients and show that mean LVM at FOS entry was higher in patients who died relative to patients who were still alive (Table 4). Conduction abnormalities were evident in 19 patients (15 male, 4 female), 15 of whom received ERT, and were present prior to the commencement of ERT in 11 (9 male) of these 15 patients. Valvular disease was recorded in 16 patients (13 male): in three males this was recorded after the start of treatment only.

Proteinuria was recorded as present at some point in time (yes/no) in 27 of the 42 patients (21 male, 6 female). Among the 22 patients in this group who received ERT, proteinuria was present prior to the commencement of ERT in 17 patients (13 males).

Absolute data were available for 23 patients showing higher proteinuria levels in patients who died than in those who were alive (Table 4). Mean proteinuria levels based on the values available were higher in patients who received ERT [females (n=4), 1521.9 mg/ 24 hrs (range, 52.5-2495.0); males (n = 17), 900.4 mg/ 24 hrs (range, 21.0-3955.0)] than in those who did not [females (n=1), 250.0 mg/ 24 hrs; males (n = 1), 970.0 mg/ 24 hrs]. Hypertension was reported in 20 (15 male) of the patients who died. Sixteen of these patients are known to have received ERT and in 13 15 cases (12 male) hypertension was present prior to the commencement of ERT. In patients who received ERT at some point, haemodialysis was reported prior to the commencement in 4 males, peritoneal dialysis in 2 males and renal transplantation in 7 males. After the start of ERT, one further male patient started haemodialysis and 5 underwent renal transplantation. Among deceased patients who never received ERT, 2 males were on haemodialysis.

Stroke was reported in 19 of the 42 patients who died (16 male, 3 female) and TIAs in 13 patients (11 male). Of the 14 stroke patients who had received ERT, 10 (9 male) experienced strokes both before and after commencement of ERT. Two males had no further strokes after the commencement of ERT, while 2 males suffered their first stroke at/after the start of ERT. The remaining patients either did not receive ERT (3 male, 2 female) or reported stroke only prior to the start of treatment (2 male).

Chronic pain was reported in 25 (21 male), and asthma in 14 (10 male) of the 42 patients who died. of patients in FOS (Table 5). Precise data on the year of death were not available; however, the majority of these deaths occurred before ERT became available in 2001.

The most common causes of death in affected male family members were renal failure (n = 50; 58% aged 21-50 years) and cardiac disease (n = 31; 71% aged 21-50 years). The cause of death was unknown in 16 male relatives, but Fabry diseaserelated causes were suspected. Affected female relatives tended to live longer than affected male relatives. The commonest causes of death in female relatives were cerebrovascular disease (n = 16; 38% aged 21-50 years, 50% aged >50 years), followed by cardiac disease (n = 12; 92% aged >50 years) and malignancy (n = 10).

Seven female relatives died as a result of renal disease. Cause of death was unknown, but the family member was known or suspected to have suffered from Fabry disease, in 16 cases.

DISCUSSION

This report describes the baseline clinical manifestations of Fabry disease in a large cohort of patients from 19 countries worldwide enrolled in the FOS database. The number of patients enrolled in FOS has increased steadily over time since the first report in 2004 [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF]. The present analysis confirmed that the major clinical manifestations of this multisystemic disease in early adulthood were pain (acroparaesthesia), angiokeratoma and renal, cardiac and cerebrovascular disease.

Gastrointestinal manifestations and sensory organ abnormalities were also common.

Although some signs and symptoms including pain and gastrointestinal disturbances were evident from an early age, end-stage organ damage was not seen in children in

FOS.

Analysis of data on causes of death among patients currently in FOS and among their relatives, suggests that the clinical course of Fabry disease may be changing.

Consistent with previously published historical data [START_REF] Macdermot | Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males[END_REF], renal failure was the most common cause of death in affected relatives of patients in FOS (31%), the majority of whom had died prior to 2001. When analysed according to sex, renal failure was the major cause of death in affected male relatives (42%) and cerebrovascular disease was the most frequently reported cause of death in affected female relatives (25%), as has been reported previously. Cardiac disease was the second most important cause of death in both affected male and affected female relatives.

Data obtained for patients enrolled in FOS whose deaths were reported between 2001 and 2007 are somewhat different. In these 42 patients, cardiac disease was the most frequent cause of death in both males (34%) and females (57%). Only three patients died from renal disease (7%). These data suggest that deaths from renal failure are becoming less frequent over time and the majority of patients are now dying from cardiac disease. This was true irrespective of whether or not patients had received ERT, although formal comparison of deaths in ERT-treated and non-ERT-treated patients was not possible because of the limited data available from the latter group.

A recent retrospective review of 447 patients (279 male, 168 female) with Fabry disease [START_REF] Schiffmann | Fabry Disease: progression of nephropathy and prevalence of cardiac and cerebrovascular events before enzyme replacement therapy[END_REF] confirms that Fabry nephropathy has a higher prevalence, occurs earlier and progresses more rapidly in the absence of ERT among males than females. This study also found a high prevalence of cardiac events (mainly arrhythmias) in 49% of males (35% females) prior to commencement of ERT; strokes/TIAs occurred in 11 and 6% of males and, 8 and 4% of females, respectively.

Evidence from our own study suggests that cardiac involvement was well established in many of the patients who died. LVH was present in almost 70% of those enrolled in FOS who died, and was present in 7 of the 9 patients who died without receiving ERT. Increased LVM, valvular heart disease, conduction disturbances and hypertension were also present prior to commencement of ERT in most of the deceased patients. It seems likely therefore that the majority of deaths attributed to cardiac causes resulted from the progression of pre-existing cardiac involvement associated with Fabry disease. However, a proportion of these cardiac deaths may be unrelated to Fabry disease, as cardiovascular disease is a major cause of death in normal adults, accounting for nearly half of all deaths in Europe (48%) [START_REF] Allender | European cardiovascular disease statistics[END_REF]. In addition, the majority of the patients who died from cardiac-related causes also had severe manifestations affecting other organ systems (primarily renal and CNS).

In the context that there is a high prevalence of renal disease, including ESRD, among patients currently in FOS, the few deaths associated with renal failure is striking.

Improvements in supportive care, greater access to dialysis facilities and improved management of hypertension may all contribute to the lower number of deaths due to renal disease. Although ERT has not been shown to be effective in reversing ESRD, several studies confirm that both agalsidase alfa and agalsidase beta can slow the decline, or perhaps even stabilize, renal function in patients with lesser degrees of renal impairment [START_REF] Schwarting | Enzyme replacement therapy and renal function in 201 patients with Fabry disease[END_REF][START_REF] Germain | Sustained, long-term renal stabilization after 54 months of agalsidase beta therapy in patients with Fabry disease[END_REF][START_REF] Feriozzi | Agalsidase Alfa Slows the Decline in Renal Function in Patients with Fabry Disease[END_REF][START_REF] West | Clinical trial summary of agalsidase alfa and kidney dysfunction in Fabry disease[END_REF]. Current guidelines for the treatment of Fabry disease emphasize the importance of angiotensin-converting enzyme inhibitors/angiotensin receptor blockade, careful management of hypertension and early initiation of ERT [START_REF] Ortiz | Recommendations and guidelines for the diagnosis and treatment of Fabry nephropathy in adults[END_REF]. As cardiac disease is now the most commonly reported cause of death, its treatment is clearly important. Studies have shown that both agalsidase alfa and beta improve cardiac structure and function in males and females with Fabry disease [START_REF] Hughes | Effects of enzyme replacement therapy on the cardiomyopathy of Anderson-Fabry disease: a randomised, doubleblind, placebo-controlled clinical trial of agalsidase alfa[END_REF][START_REF] Spinelli | Enzyme replacement therapy with agalsidase beta improves cardiac involvement in Fabry's disease[END_REF][START_REF] Weidemann | Improvement of cardiac function during enzyme replacement therapy in patients with Fabry disease: a prospective strain rate imaging study[END_REF][START_REF] Sachdev | Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy[END_REF]. If treatment of cardiac and renal dysfunction are optimized and patients receive optimal supportive care, it is also conceivable that early stroke may be prevented, although recent reports suggest that premature stroke may still occur in patients receiving ERT [START_REF] Clarke | Narrative review: Fabry disease[END_REF]. Unfortunately, due to the lack of an appropriate control group, it was not possible to evaluate specifically whether ERT changed the progression of disease manifestations or increased the life expectancy of patients with Fabry disease in the present study.

As is generally the case for most non-interventional observational databases, there are several limitations to this study. First, as all clinical decisions and data entry are at the discretion of the attending physician, data sets are often incomplete and there is a lack of standardization in the assessments conducted between patients. There is also variability between patients in terms of the treatment protocols used and the supportive care provided. Thirdly, patient-reported data on the causes of death in relatives are subject to selective recall and these data have not been verified with reference to source documents. In addition, only simple data are available for some variables, such as cause of death. Finally, the nature of the database makes it likely that untreated and mildly affected patients are under reported.

In conclusion, these data suggest that the natural clinical course of Fabry disease may be changing. The importance of renal failure as a cause of death is decreasing, while the importance of cardiac disease is increasing. This is partly a result of significant improvements in supportive care, particularly for renal failure and hypertension, which have occurred in recent years. Such improvements, in addition to the provision of ERT, may be contributing to changes in morbidity and mortality associated with Fabry disease. In future, it is hoped that these natural history data will enable the accurate assessment of the impact of ERT in patients with Fabry disease. There remains, however, a very real need for new treatments, particularly for oral therapies and for the development of treatment approaches that are better able to access the CNS and organs such as the heart and kidney.

FOS

  has been approved by the Ethics Institution Review Boards of participating centres (Appendix A), and all patients gave written informed consent. To minimize bias, data from all consenting patients are entered. All patients enrolled in FOS are receiving, or are candidates for, ERT with agalsidase alfa. On enrolment, each patient's medical history is documented by a physician or nurse specialist, including the year of diagnosis of Fabry disease, signs and symptoms of the disease, treatment, demographic details and family history. All measurements performed routinely in clinical practice are entered into the database. Assessments of cardiac and renal function, ophthalmological, gastrointestinal and audiological examinations are optional. Anonymized data are submitted electronically by participating physicians to the central FOS database. Disease severity is systematically categorized in all patients

Table 1 .

 1 Mean baseline characteristics of the 1453 patients with Fabry disease in the

	vs. 11.0 (9.0), respectively;

Signs and symptoms in male and female patients The

  prevalence and age at onset of the main signs and symptoms of Fabry disease in males and females at entry into FOS are shown in Table2. As in the previous analysis

	of FOS data, the most frequently reported signs and symptoms of Fabry disease were
	neurological, affecting 75% of males and 61% of females (Table 2). Cardiac, ocular,
	gastrointestinal, dermatological, auditory and renal manifestations were also common.
	Renal signs and symptoms of Fabry disease were reported in 693 patients in FOS.
	Proteinuria was the most common renal manifestation, being observed in 332 males
	and 246 females. Renal failure was present in 134 males and 21 females. In total, 89

males and five females had end-stage renal disease (ESRD) [mean age of onset, 32.3 (19.7) and 23.3 (21.3) years, respectively]. Cardiac manifestations were recorded in 422 male and 376 female patients. Of these patients, 284 males and 180 females had left ventricular hypertrophy (LVH), which was reported to have begun at 28.7 (19.3) and 34.1 (24.9) years of age, respectively. Cerebrovascular events occurred with approximately the same frequency in males and females [25% (n = 172) and 21% (n = 159), respectively]. Stroke was reported in 39 females and 63 males [mean age of onset, 51.4 (14.1) and 39.2 (11.5), respectively]. TIA was recorded in 46 females and 60 males [mean age of onset, 46.6 (14.8) and 38.4 (13.6), respectively].

Table 2 .

 2 Prevalence and age at onset of organ system involvement in patients with Fabry disease enrolled in the Fabry Outcome Survey.

		Males (n = 699)	Females (n = 754)	Total (n = 1453)
	Organ system	Percentage with signs/	Mean age at onset	Percentage with signs/	Mean age at onset	Percentage with signs/	Mean age at onset
		symptoms	(years)	symptoms	(years)	symptoms	(years)
	Neurological	75	15.1 (15.0)	61	20.9 (17.9)		17.9 (16.6)
	Cardiac	60	29.2 (14.4)	50	34.5 (17.6)		31.6 (16.1)
	Ocular	58	29.2 (15.1)	49	31.9 (18.3)		30.5 (16.8)
	Gastrointestinal	57	22.2 (16.1)	45	26.8 (19.2)		24.3 (17.7)
	Dermatological	66	20.0 (15.4)	37	30.8 (19.0)		24.3 (17.7)
	General	59	18.7 (15.9)	44	26.1 (19.0)		21.8 (17.7)
	Auditory	56	29.0 (15.3)	44	33.3 (19.1)		31.0 (17.2)
	Renal	59	32.6 (12.7)	38	38.5 (16.2)		35.0 (14.5)
	Musculo-skeletal	39	26.9 (16.8)	34	34.7 (18.4)		30.5 (17.9)
	Vascular	35	34.5 (12.9)	25	41.7 (16.4)		37.4 (14.9)
	Psychiatric	27	32.4 (15.3)	23	36.5 (17.0)		34.4 (16.2)
	Respiratory	29	32.9 (17.3)	21	36.3 (20.5)		34.3 (18.7)
	Cerebrovascular	25	31.3 (15.2)	21	36.4 (18.1)		33.7 (16.8)
	Endocrinological	5	31.4 (20.4)	8	42.1 (17.9)	7	39.0 (19.1)
	Genital	6	35.0 (17.9)	1	38.6 (16.7)	4	35.7 (17.5)

respectively. The primary cause of death in both male and female patients was cardiac disease, reported in 34% (n = 12) and 57% (n = 4) of cases, respectively. Of the 42 patients who died, 33 (28 males) had received ERT at some point whilst alive: and among them cardiac disease remained the most common cause of death (n = 12; 36%), followed by infection (n = 5; 15%) and multisystemic disease (n = 4; 12%).

Renal failure was responsible for three deaths (n = 3; 9%). Cardiac disease was also the most common cause of death (n = 4 of 9; 44%) among patients who died but had not received ERT at any point. However, data on deaths in patients not receiving ERT have only been collected since August 2006, and therefore the untreated group is not entirely representative of all untreated patients.

Table 3 .

 3 Cause of death in affected male and female patients in the Fabry Outcome Survey. Data on patients who had received ERT at some point prior to death were available during the period between 2001 and 2007. Information on deaths of patients who never received ERT could be added from August 2006 onwards.

	Cause of death Received ERT at some time	Never received ERT		Overall
		Male	Female	Total	Male	Female	Total	Male	Female	Total
	Cardiac	10	2	12	2	2	4	12	4	16
	Renal	3	0	3	0	0	0	3	0	3
	Cerebrovascular	3	0	3	1	0	1	4	0	4
	Respiratory	2	0	2	1	0	1	3	0	3
	Malignancy	1	1	2	1	0	1	2	1	3
	Infection	5	0	5	0	0	0	5	0	5
	Multisystemic	2	2	4	2	0	2	4	2	6
	Other	2	0	2	0	0	0	2	0	2
	Total	28	5	33	7	2	9	35	7	42

Table 4 .

 4 Mean (SD) data at FOS entry according to whether patients were alive or dead at the time of the analysis.

	Adult females

Table 5 .

 5 Number of deaths in relatives of patients enrolled in the Fabry Outcome Survey reported up to December 2007. Data are shown according to reported age at death.

	Cause

of death Number of deaths in male relatives Number of deaths in female relatives Overall number of deaths

		Age at death (years)				Age at death (years)				Age at death (years)
	0-20	21-50	>50	Unknown	Total	0-20	21-50	>50	Unknown	Total	0-20	21-50	>50	Unknown Total
	Cardiac	22	8	1	31	0	1	11	0	12	0	23	19	1	43
	Renal	29	14	7	50	0	5	2	0	7	0	34	16	7	57
	Cerebrovascular	5	5	2	12	0	6	8	2	16	0	11	13	4	28
	Respiratory	0	1	0	1	0	0	0	0	0	0	0	1	0	1
	Malignancy	2	1	0	3	0	4	5	1	10	0	6	6	1	13
	Infection	3	0	2	5	0	0	1	0	1	0	3	1	2	6
	Multisystemic	0	0	0	0	0	0	1	0	1	0	0	1	0	1
	Other	7	3	6	16	0	4	5	7	16	0	11	8	13	32
	Total	68	32	18	118	0	20	33	10	63	0	88	65	28	181

* p<0.001 compared with males.

ACKNOWLEDGEMENTS

All FOS Investigators who have submitted data from their patients to the FOS database (see www.outcomesurveys.com for complete list of contributors). The FOS database is under the independent control of the FOS International Board. Editorial assistance to the authors was provided by Dr Harriet Crofts (Oxford PharmaGenesis Ltd., Oxford, UK).

Data collection and analysis in FOS are supported by Shire HGT, Danderyd, Sweden.

The sponsor had no role in the interpretation of data or writing of the report.

COMPETING INTERESTS

AM received speaker fees, research funds and funding for symposium attendance from Shire Human Genetic Therapies (HGT); JTRC received funding for symposium attendance from Shire HGT; RG received speaker fees and funding for symposium attendance from Shire HGT; PE received speaker fees, funding for symposium attendance and unrestricted educational grants from Shire HGT; AL and GSP received speaker fees, travel grants and research support from both Shire HGT and Genzyme; MB received speaker fees, funding for symposium attendance and unrestricted scientific grants from Shire HGT, Genzyme and Biomarin. Honoraria were not paid in relation to FOS data entry or for the writing of this report.

FUNDING Ethics committee approval

The FOS database has been approved by the Ethics Institution Review Boards of the over 100 participating centres, and all patients gave written informed consent. As so many investigators and participating centres are involved in FOS, we have provided a list of the investigators who submitted data to FOS (correct as of December 2007), together with their location:

LICENCE STATEMENT

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