This electromagnetic system can be described by the following general flux-currents relationships:

Φ sx = L sx i sx + M srx i rx Φ rx = L rx i rx + M rsx i sx ( 1
)
where x is put for d or q, and the cyclic inductances are functions of the currents (i sd , i sq , i rd , i rq ).

The curves of cyclic inductances can be obtained by injecting only one current: L sx = Φ sx /i sx , L rx = Φ rx /i rx , M srx = Φ sx /i rx and M rsx = Φ rx /i sx .

In paper [5], we demonstrated that the magnetic state of the induction machine depends on the modulus of the magnetizing current vector |I m | = (i sd + αi α rd ) 2 + (i sq + αi α rq ) 2 , where α is the reference factor. Its expression is given in [START_REF] Semail | Modélisation et réalisation d'un actionneur asynchrone et de sa commande vectorielle[END_REF] and it does not depend on the magnetic state of the machine in the contrary of the factor L s /M which includes the leakage inductance in the numerator that varies with the magnetic state.

The established saturated model can be described by the following electromechanical equations:

electrical equations:

V s = R s I s + dΦ s dt + j dΨ dt Φ s 0 = R α r I α r + dΦ α r dt + j d(Ψ -pθ) dt Φ α r (2)
flux-current relationships:

Φ s = L s I m I s + M α I m I α r Φ α r = L α r I m I α r + M α I m I s (3)
mechanical equation:

j dΩ dt = C em -fΩ -C r . ( 4
)
The index α is adopted for all rotor electrical parameters to show that this model is established for a reference factor α.

Because of the fact that leakage inductances are included in L s and L r , this model was called the two-phase saturated model of separated leakage inductances.

From equation (3) we can see that the saturation cross effect is introduced in the cyclic inductances evolutions. For example, the stator flux of the D axis does not vary only with i sd and i rd , but also with i sq and i rq because L s and M vary with I m .

Electrical parameters shown from the stator

In this section we are going to find the electrical parameters that can experimentally measured from the stator at several saturation levels.

First of all, from the saturated model described before, we will find an equivalent saturated model with a unity reference factor, α = 1. This can easily be done by introducing the following new parameters in the electrical equations and in the flux-currents relationships:

Φ r = Φ α r /α, I r = αI α r , M = M α /α, L r = L α r /α 2 and R r = R α r /α 2
, so we obtain the equivalent model described by the following electromechanical equations:

electrical equations:

V s = R s I s + dΦ s dt + j dΨ dt Φ s 0 = R r I r + dΦ r dt + j d(Ψ -pθ) dt Φ r (5)
flux-current relationships:

Φ s = L s I m I s + M I m I r Φ r = L r I m I r + M I m I s (6)
mechanical equation:

j dΩ dt = C em -fΩ -C r (7)
where I m = (i sd + i rd ) 2 + (i sq + i rq ) 2 is the modulus of the magnetising current vector.

Figure 2 shows the electrical circuit of this equivalent model. For a certain saturation level s, the cyclic inductances do not vary. So, If we apply the 2 to 3 inverse Park transformation for Ψ = 0 in the stator, and for Ψ = pθ in the rotor, we obtain the following electrical equations of each phase:

L M L s R r R s V s I r I s r j d(Ψ-pθ) dt Φ r dΨ dt Φ s j
[V s ] 3 = R s [I s ] 3 + L s s d[I s ] 3 dt + M s d[I r ] 3 dt [0] 3 = R r [I r ] 3 + M s d[I s ] 3 dt + L s r d[I r ] 3 dt where [X] t 3 = [x a , x b , x c ].
For the a stator phase and the a rotor phase, and for a sinusoidal steady state s, we can write:

V s = R s I s + jω s (L s s I s + M s I r ) 0 = R r g I r + jω s (M s I s + L s r I r )
where:

V s = V s I s = I s e -jζ1 , ζ 1 = ∠(V s , I s ) I r = I r e -jζ2 , ζ 2 = ∠(V s , I r )
g is the slip. The equivalent circuit is shown in Figure 3. To obtain the electrical parameters shown from the stator, we introduce the magnetizing stator current I ms = I s + M s L s s I r and the rotor current shown from the stator

I r = M s L s s I r (Fig. 4): V s = R s I s + jω s L s s I ms 0 = R r g I r + jω s (L s s I s + N s I r )
where

N s = L s s L s s L s r (M s ) 2 -1 is the leakage inductance to- taled in the rotor, and R s r = L s s M s 2
R r is the rotor resistance shown from the stator which depends on the magnetic state of the machine.

We call the model presented in Figure 4 the steady state model with leakage inductance totaled in the rotor for a certain saturation level. For each magnitude of the stator voltage, the value of L si s corresponding to the steady state s i is calculated using the following expression:

L si s I si m = 3(E s rms ) 2 ω s Q si ( 8
)
where Q is the reactive power. 3-To calculate rotor parameters shown from the stator for several saturation level, we propose to do several experimental measurements at a small slip and at a stator frequency smaller than the nominal one. The reason of these conditions is that the induction machine can not be saturated if we apply the classical measurement method when the rotor is blocked. To demonstrate this idea, we develop the following study using the cyclic inductances curves obtained for a 4 KW induction machine and calculated by a finite elements calculation program [5,[START_REF] Kasmieh | Modélisation et caractérisation de la saturation magnétique des machines asynchrones en vue de la commande[END_REF]. From Figure 4 and for a steady state s i we have:

V si s = R s I si s + Z si Ls I si ms ( 9
)
I si ms = 1 + L si s M si -1 Z si Ls Z si rotor -1 I si m ( 10
)
I si s = 1 - Z si Ls Z si rotor 1 + L si s M si -1 Z si Ls Z si rotor -1 I si m ( 11
)
where Z Ls is the impedance of L s and Z rotor is the impedance of the rotor circuit.

From these previous equations we obtain Figures 6 and7.

We notice, from these figures, that it is very difficult to saturate the machine for high values of the slip and the stator frequency.

To illustrate the previous study, we show in Figure 8 two simulated dynamic responses of the 4 KW machine for a slip of 100% and for 10%. It is clear that the machine can be easily saturated if the value of the slip is small. Therefore, to calculate R r and N for several saturation levels, we have to do measurements at a small value of the slip to obtain a weak value of the rotor current shown from the stator which opposes to the stator current, and at a stator frequency smaller than the nominal frequency to have a small impedance of the inductance L s .

For each steady state s i , determined by the magnitude of the stator voltage V si s , and for a fixed stator frequency, the values of R r and N are calculated from Figure 4 using the following expressions:

R si r = g si P si 1 3(E si eff ) 2 (P si 1 ) 2 + (Q si 1 ) 2
(12)

N si = Q si 1 ω s 3(E si eff ) 2 (P si 1 ) 2 + (Q si 1 ) 2
(13)

P si 1 = P si -3(I si seff ) 2 R s (14) Q si 1 = Q si - 3(E si eff ) 2 ω s L si s ( 15
)
where P and Q are the active and the reactive powers.

From equation ( 15) we notice that we have to know the value of the stator cyclic inductance for each experiment. This value can be calculated by doing the approximation I si ms ≈ I si m (small slip) and by using the approximated equation:

E si ≈ 2 3 ω s L si s I si m . ( 16
)
So, for each experiment we measure E si and we search the intersection point between the curve L si s I si m , found by doing the no load experiments, and 3 2 E s i ωs .

Application of the new experimental measurement method on a 4 KW squirrel cage induction machine

We apply the new measurement method on a machine of 4 KW. This machine is loaded with a velocity controlled synchronous machine, Figure 9.

The stator frequency can be changed using a PWM inverter.

The measurements are done at a stator frequency of 20 Hz, and the measurements of the rotor parameters shown from the stator are done at a slip of 10%. The results obtained by the new experimental measurement method are presented in Figures 10 and11.

The evolution obtained of the inductance L s with these measurements is very close to the evolution calculated with a finite elements calculation program.

From Figure 11, and for the measurements at a small slip of rotor parameters shown from the stator, we notice that the values of the total leakage inductance N experimentally measured are bigger than those calculated using a finite elements calculation program. This can be explained by the fact that the finite elements calculation program is a two-dimension program and it does not take in to account all leakage inductances.

Here, we mention that this method has a weak sensitivity in measuring several points when the machine starts to be saturated, Figure 10. This is because of the big variation of the cyclic inductances for a small variation of the stator voltage. This is why we obtain an irregular curve in this zone.

Establishment of a new dynamic saturated model of the induction machine

Now, we will find another two-phase saturated model that only needs the electrical parameters shown from the stator.

From equation ( 6) we can write the stator flux vector as follows:

Φ s = L s I ms (17)
where I ms = I s + M Ls I r = I s + I r is the magnetizing stator current vector.

We add and substrate M 2 Ls I r from the expression of the rotor flux vector, then we multiply it by Ls M :

Φ r = (L s + N )I r + L s I s (18)
where Φ r = Ls M Φ r and N = L s LsLr M 2 -1 is the leakage inductance totaled in the rotor.

If we introduce the new vectors Φ r and I r in the electrical equations ( 5), we obtain a new two-phase saturated model that can be described by: electrical equations:

V s = R s I s + dΦ s dt + j dΨ dt Φ s 0 = R r I r + dΦ r dt + 1 Γ dΓ dt Φ r + j d(Ψ -pθ) dt Φ r ( 19
)
where Γ = M Ls flux-current relationships:

Φ s = L s I ms Φ r = (L s + N )I r + L s I s (20)
mechanical equation:

j dΩ dt = C em -fΩ -C r . ( 21
)
The electrical circuit of this model is shown in Figure 12.

To resolve the electromechanical equations of this new model, we have to do the approximation Γ ≈ Ls Ls+N and apply the same algorithm used to solve the electromechanical equations of the two-phase saturated model of separated leakage inductances which we presented in paper [5].

Experimental validation

Figure 13 shows the real dynamic responses of the 4 KW machine (curves 1) with the dynamic responses of the The experiments are done at a maximum magnitude of the stator voltages of V s = 40 V, and at a stator voltages frequency of f s = 5 Hz. So, under these conditions, we are sure that the machine has a greater saturation level than the nominal saturation level which is given at V s = 311 V and at f s = 50 Hz. We can also see from the previous figure that the stator cyclic inductance is 23.8% smaller than its linear value.

Remarks

In this paper we do not take into account the iron losses. This can be introduced by adding a resistance R i in parallel with L s . We can measure this resistance while doing the second step of the new experimental measurement method using the expression:

R i = 3E 2 eff p -3R s I 2 seff
in this case, the electrical equations (19) stay the same, but the cyclic inductances vary with I m = I s + I r where I s = I s -I si and I si is the current of the resistance R i , Figure 14.

Conclusion

In this paper we presented a new experimental measurement method of the electrical parameters of the induction machine shown from the stator. This method was applied on a 4 KW induction machine and validated by comparing its results with the results obtained by a finite elements calculation program. The aim of searching such a method is to find a new two-phase saturated model that uses the electrical parameters measured from the stator.

This model was called the two-phase saturated model with leakage inductance totaled in the rotor.

The study has a very big advantage from the industrial point of view, especially for a squirrel cage induction machine. It avoids the use of a finite elements calculation program to calculate the evolution of cyclic inductances with the variation of saturation level in the air gap. By doing the proposed experiments, which are relatively simple, we can find the evolutions of the electrical parameters of the machine shown from the stator and then use the new two-phase saturated model with leakage inductance totaled in the rotor.

Fig. 1 .

 1 Fig. 1. Two-phase electromagnetic system of the induction machine.

Fig. 2 .

 2 Fig. 2. Equivalent saturated model with a unity reference factor.

Fig. 3 .

 3 Fig. 3. Equivalent circuit of the induction machine for a certain saturation level.

Fig. 4 .

 4 Fig. 4. Steady state model with leakage inductance totaled in the rotor.

Fig. 5 .

 5 Fig. 5. No-load circuit of the induction machine.

Fig. 8 .

 8 Fig. 8. Simulation example for a small slip and for a blocked rotor.

Fig. 9 .

 9 Fig. 9. Electromechanical system used to measure the electrical parameters shown from the stator.

Fig. 10 .

 10 Fig. 10. Results of no load experiments.

Fig. 11 .Fig. 12 .

 1112 Fig. 11. Results of small slip experiments.

Fig. 13 .Fig. 14 .

 1314 Fig. 13. Dynamic responses of the 4 KW machine. (1) Real response. (2) Saturated model. (3) Linear model.

4 New experimental measurement method of electrical parameters of the steady state model with leakage inductance totaled in the rotor for several saturation levels

Now, we will search a new experimental measurement method of the electrical parameters of the circuit shown in Figure

4

for several saturation levels s.

1-The stator resistance R s does not depend on the magnetic state of the machine, so we can measure it by applying a continuous stator voltage on one of the stator phases. 2-To measure L s we propose to do several no-load measurements for several amplitudes of the stator voltage and at a fixed frequency smaller than the nominal frequency. The circuit employed in this case is the following:


Luisa Laureti

Lara Lee

Gaëlle Philippin

Michel Kahi

Vincent Pagès
email: vincent.pages@cnrs.fr

Luisa Laure6
email: luisa.lauree@inserm.fr

Extending the gap and loading RecA: the presynaptic phase plays a pivotal role in modulating lesion tolerance pathways

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The genome of all living organisms is constantly damaged and some lesions can impair replication, potentially leading to mutagenesis and genome instability. In the presence of an unrepaired replication-blocking lesion, a single-stranded DNA (ssDNA) gap is formed, due to the initiation of the next Okazaki fragment (on the lagging strand) or to a repriming event (on the leading strand) [START_REF] Rupp | Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation[END_REF][START_REF] Lopes | Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions[END_REF][START_REF] Heller | Replication fork reactivation downstream of a blocked nascent leading strand[END_REF]. In order to complete replication and preserve cell survival, the ssDNA gap is filled in by one of the two lesion tolerance pathways, identified both in prokaryotes and eukaryotes: 1) Translesion Synthesis (TLS), which employs specialized DNA polymerases able to replicate the damaged DNA, with the potential to introduce mutations (4-6); 2) Damage Avoidance (DA) pathways, which use the information of the sister chromatid to bypass the lesion in a non-mutagenic way through homologous recombination mechanisms [START_REF] Prado | Homologous recombination maintenance of genome integrity during DNA damage tolerance[END_REF]. In Escherichia coli, we have shown that in nonstressed conditions Homology Directed Gap Repair (HDGR), a DA mechanism that relies on the recombinase RecA, is the major lesion tolerance pathway employed by cells while TLS pathway is a minor one [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF][START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF]. We have also identified another lesion tolerance strategy, named Damaged Chromatid Loss (DCL) that promotes cell proliferation at the expense of losing the damaged chromatid; in particular when HDGR mechanism is impaired, cells can survive by replicating only the undamaged chromatid [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF].

In E. coli there are two well-known RecA dependent recombinational pathways: the RecBCD pathway, involved in the repair of double strand breaks, and the RecF pathway (also known as the RecFOR pathway), involved in the repair of ssDNA gap and required for an efficient SOS induction (reviewed in [START_REF] Smith | Homologous recombination in procaryotes[END_REF][START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF][START_REF] Spies | Homologous recombination by RecBCD and RecF pathways. The bacterial chromosome[END_REF]. Originally, the RecF pathway has received less attention because most of the studies on recombination focused on processes initiated by a double strand break, such as conjugation or transduction. However, in the last decades, it became clear that this pathway, other than be a "back-up" recombinational pathway in the absence of RecBCD, plays an important role during replication of a damaged DNA. Recently, it was shown that during non-stressed conditions, homologous recombination (RecA and RecF dependent) is mainly required to repair ssDNA gaps during replication other than double strand breaks [START_REF] Xia | Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase[END_REF].

The RecF pathway is evolutionary conserved in all bacteria [START_REF] Rocha | Comparative and evolutionary analysis of the bacterial homologous recombination systems[END_REF] and functional orthologs are also found in Eukaria. This pathway encompasses several proteins and is divided into three distinct phases [START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF]: i) the presynaptic phase in which the damaged ssDNA gap is processed by different proteins (i.e. SSB, RecF, RecO, RecR, RecJ, RecQ) to promote the formation of an active RecA nucleofilament; ii) the synaptic phase in which homology pairing and strand exchange by the RecA nucleofilament occur to produce a D-loop and iii) the postsynaptic phase involving the processing of the recombinational intermediates by the resolvase RuvABC or by the helicase RecG. Some of these proteins have already been well characterized in vitro [START_REF] Kowalczykowski | Biochemistry of homologous recombination in Escherichia coli[END_REF][START_REF] Handa | Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli[END_REF][START_REF] Morimatsu | RecFOR proteins target RecA protein to a DNA gap with either DNA or RNA at the 5′ terminus: implication for repair of stalled replication forks[END_REF][START_REF] Morimatsu | RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination[END_REF], but their role in vivo and their impact on lesion tolerance pathways is not yet fully elucidated. The presynaptic phase is of particular interest since the ssDNA gap is the common substrate for both HDGR and TLS, hence how the ssDNA gap is processed to produce a RecA nucleofilament might influence the choice of lesion tolerance pathways.

Several genetic analyses indicate that the recF, recO and recR genes belong to the same epistasis group as the double mutants behave like the single mutants, at least for the UV sensitivity and the delay in SOS induction [START_REF] Tseng | Involvement of RecF pathway recombination genes in postreplication repair in UVirradiated Escherichia coli cells[END_REF][START_REF] Whitby | Altered SOS induction associated with mutations in recF, recO and recR[END_REF][START_REF] Thoms | Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant[END_REF](22)[START_REF] Morimatsu | RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair[END_REF]. According to in vivo and in vitro studies, it was proposed that two complexes, RecOR and RecFR, are formed whose function at a ssDNA gap is to disassemble the filament of single strand binding (SSB) proteins in order to load the recombinase RecA and promote homologous recombination (22,[START_REF] Morimatsu | RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair[END_REF]. In vitro, high amounts of the RecOR complex are sufficient to displace SSB proteins and load RecA, promoting strand exchange in the absence of RecF [START_REF] Umezu | Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein[END_REF][START_REF] Sakai | RecFOR and RecOR as distinct RecA loading pathways[END_REF]. On the other hand, RecF was shown to bind to gapped substrates, preferentially on the 5' double strand-single strand (ds-ss) DNA junction [START_REF] Morimatsu | RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair[END_REF][START_REF] Hegde | Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate[END_REF] and often colocalize with the replisome [START_REF] Henrikus | RecFOR epistasis group: RecF and RecO have distinct localizations and functions in Escherichia coli[END_REF]. The UV sensitivity phenotype of the recFOR deficient strains is presumably due to a defect in RecA loading that leads to recombinational repair deficiency. Accordingly, a RecA mutant (i.e., the recA730 allele) able to load itself on a ssDNA partially restores UV resistance of the recFOR deleted strains [START_REF] Lavery | Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins[END_REF][START_REF] Wang | Cosuppression of recF, recR and recO mutations by mutant recA alleles in Escherichia coli cells[END_REF].

The product of the recJ gene has also been associated with the RecF pathway [START_REF] Lloyd | Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair[END_REF]; however, compared to the recFOR deficient strains, no strong UV sensitivity phenotype was observed when the recJ gene was deleted [START_REF] Tseng | Involvement of RecF pathway recombination genes in postreplication repair in UVirradiated Escherichia coli cells[END_REF]. Later, Courcelle and Hanawalt (31,32) proposed that the products of recJ and recQ genes were involved in the repair of ssDNA gaps by processing the nascent lagging DNA strand during replication blockage. The helicase RecQ (3'->5') would help the exonuclease RecJ (5'->3') to enlarge the ssDNA at the 5' end of the gap to lengthen the substrate for RecA filament formation. In vitro data showing that SSB protein is able to stimulate the activity of RecQ and of RecJ corroborate the hypothesis that both RecQ and RecJ are recruited at the ssDNA gap formed upon lesion encountering [START_REF] Morimatsu | RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination[END_REF]. Recently, we have shown that in the presence of an unrepaired lesion, RecJ is required to enlarge the ssDNA gap not only in the lagging strand but also in the leading strand [START_REF] Chrabaszcz | DNA lesions proximity modulates damage tolerance pathways in Escherichia coli[END_REF], thus providing further evidence of the in vivo role of RecJ in the presynaptic phase.

In the present study, we have explored the role of the genes involved in the presynaptic phase of the RecF pathway, their genetic interaction and their effect on lesion tolerance pathways. We confirmed that, regarding lesion bypass, the recF, recO and recR genes belong to same epistasis group, likewise recJ and recQ genes showed to be epistatic. Defect in RecA loading (mediated by the RecFOR complexes) or in enlarging the ssDNA gap (mediated by RecQ and RecJ) results in HDGR impairment that is partially compensated by TLS independently of SOS induction. Interestingly, our data revealed a strong genetic interaction between recF and recJ (as well as recF and recQ), showing an additive effect of the deletion of both genes. Indeed, loss of these two genes results in a recA deficient-like phenotype in which HDGR is almost completely abolished, indicating that both the size of the ssDNA gap and the loading of RecA play an important role in the formation of an efficient Dloop structure, thus favoring the non-mutagenic HDGR mechanism over TLS pathway.

MATERIAL AND METHODS

Bacterial strains and growth conditions

All E. coli strains used in this work are listed in Supplementary Table 1. They are either derivative of strains FBG151 and FBG152 [START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF], that carry a plasmid that allows the expression of the int-xis genes after IPTG induction, or derivative of strains EC1 and EC2 (this study), in which the int-xis genes have been inserted into the chromosome of E. coli. Strains were grown on solid and in liquid Lysogeny Broth (LB) medium. Gene disruptions were achieved by the one-step PCR method [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF] or by P1 transduction using the Keio collection [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection[END_REF]. Following the site-specific recombination reaction, the lesion is located either in the lagging strand (FBG151 or EC1 derived strains) or in the leading strand (FBG152 or EC2 derived strains). Antibiotics were used at the following concentrations: ampicillin 50 or 100 μg/ml; tetracycline 10 μg/ml, kanamycin 100 μg/ml, chloramphenicol 30 μg/ml. When necessary, IPTG and X-Gal were added to the medium at 0.2mM and 80 μg/ml, respectively. Plasmids pVP135 expresses the integrase and excisionase (int-xis) genes from phage lambda under the control of a trc promoter that has been weakened by mutations in the -35 and the -10 region. Transcription from Ptrc is regulated by the lac repressor, supplied by a copy of lacI q on the plasmid. The vector has been modified as previously described [START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF]. pVP146 is derived from pACYC184 plasmid where the chloramphenicol resistance gene has been deleted. This vector, which carries only the tetracycline resistance gene, serves as an internal control for transformation efficiency. pVP141-144 and pGP1, 2 and 9 are derived from pLDR9-attL-lacZ as described in [START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF]. pLL1, pLL2c and pLL7 are derived from pVP141 and contain several genetic markers as previously described [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF]. All these plasmid vectors contain the following characteristics: the ampicillin resistance gene, the R6K replication origin that allows plasmid replication only if the recipient strain carries the pir gene, and the 5' end of the lacZ gene in fusion with the attL site-specific recombination site of phage lambda. The P'3 site of attL has been mutated (AATCATTAT to AATTATTAT) to avoid the excision of the plasmid once integrated. These plasmids are produced in strain EC100D pir-116 ( f r o m E p i c e n t r e B i o t e c h n o l o g i e s , c a t # EC6P0950H) in which the pir-116 allele supports higher copy number of R6K origin plasmids. Vectors carrying a single lesion for integration were constructed as previously described following t h e g a p -d u p l e x m e t h o d [START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF] . A 1 3 -m e r oligonucleotide, 5′-GCAAGTTAACACG-3′, containing no lesion or a TT(6-4) lesion (underlined) in the HincII site was inserted either into the gapped-duplex pLL1/2c leading to an out of frame lacZ gene (to measure HDGR) or into the gapped-duplex pGP1/2 leading to an in frame lacZ gene (to measure TLS0). A 15-mer oligonucleotide 5'-ATCACCGGCGCCACA-3' containing or not a single G-AAF adduct (underlined) in the NarI site was inserted into the gapped-duplex pLL1/7 (to measure HDGR) or into the gapped-duplexes pVP141-142 or pVP143-144 to score respectively for TLS0 Pol V-dependent and for TLS-2 Pol IIdependent. A 13-mer oligonucleotide, 5′-GAAGACCTGCAGG, containing no lesion or a dG-BaP(-) lesion (underlined) was inserted into the gapped-duplex pVP143/pGP9 leading to an in frame lacZ gene (to measure TLS).

Monitoring HDGR and TLS events

The protocol for lesion integration assay is described in details in [START_REF] Laureti | A non-catalytic role of RecBCD in homology directed gap repair and t r a n s l e s i o n s y n t h e s i s . N u c l e i c A c i d s R e s e a r c h[END_REF]. Cells were diluted and plated before the first cell division using the automatic serial diluter and plater EasySpiral Dilute (Interscience) and were counted using the Scan 1200 automatic colony counter (Interscience). No differences were observed when we used FBG or EC derivative strains. Following the integration of the pLL1/2c vector (TT6-4 lesion) or pLL1/7 vector (AAF lesion), sectored blue/white colonies represent HDGR events. Following integration of the pVP141/142, pVP143/144, pGP1/2, pVP143/pGP9 vectors (AAF, TT6-4 or BaP lesion, respectively), sectored blue/white colonies represent TLS events. The relative integration efficiencies of lesion-carrying vectors compared with their lesion-free homologues, normalized by the transformation efficiency of pVP146 plasmid in the same electroporation experiment, allow the overall rate of lesion tolerance to be measured. For each lesion we combine the results obtained with the different plasmids to measure HDRG and TLS events (for BaP lesion we only measure TLS events). The data in every graph represent the average and standard deviation of at least three independent experiments of a lesion inserted in the leading (or in the lagging) orientation. Tolerance events (Y axis) represent the percentage of cells able to survive in presence of the integrated lesion compared to the lesion-free control.

RESULTS

In the present work, we set out to investigate the presynaptic phase of the RecF pathway during 4 A) The system is based on the phage lambda site-specific recombinaEon and it requires a recipient E. coli strain with a single a)R site fused to the 3'-end of the lacZ gene and a non-replicaEng plasmid construct containing a single DNA lesion (red triangle) in the 5'-end of the lacZ gene fused to the a)L site. The recombinaEon reacEon between a)L and a)R is controlled by ectopic expression of phage lambda int-xis proteins (provided by pVP135), and leads to the integraEon of the lesion-containing vector into the chromosome. Integrants are selected on the basis of their resistance to ampicillin and the chromosomal integraEon restores an enEre lacZ gene. B) Plasmid duplexes used to monitor TLS events (see also [START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF]. The non-damaged strand (A+B markers) contains a short sequence heterology opposite the lesion that inacEvates the lacZ gene and serves as a geneEc marker for strand discriminaEon. Only the replicaEon by TLS polymerases of the A+C markers will give rise to a funcEonal lacZ gene and therefore to a sectored blue-white colony. For the G-AAF lesion, one construct measures TLS0 events specific of Pol V-dependent TLS events, while another one contains a +2 frameshit that allows to measure TLS-2 events specific of Pol II when the G-AAF lesion is inserted in the NarI hotspot sequence (see [START_REF] Pagès | How DNA lesions are turned into mutations within cells?[END_REF]. C) Plasmid duplexes used to specifically monitor strand exchange mechanisms, i.e. HDGR events (see also LaureE et al., 2015). The system is a modified version of the plasmid constructs used to monitor TLS events. In these plasmids four geneEc markers have been designed in order to disEnguish the replicaEon of the non-damaged strand (containing markers A and B) from the damaged strand (containing markers C and D). Using a combinaEon of frameshia and stop codon, we inacEvated lacZ gene on both the damaged (C-D) and undamaged (A-B) strands of the vector. Only a strand exchange mechanism by which replicaEon has been iniEated on the damaged strand (incorporaEon of marker C), and where a template switch occurred at the lesion site (leading to incorporaEon of marker B) will restore a funcEonal lacZ gene (the combinaEon of markers C and B contains neither a stop codon nor a frameshia); therefore leading to sectored bluewhite colonies. When the damaged strand is loss, only the A and B marker will be replicaEng giving rise to a white colony. the encounter of a replication fork with a DNA lesion. We used a previously described assay that allows to insert a single replication-blocking lesion into the chromosome of E. coli and monitor lesion tolerance mechanisms by a colorimetric assay based on the lacZ reporter gene [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF][START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF][START_REF] Pagès | Inserting site-specific DNA lesions into whole genomes[END_REF]. Briefly, a non-replicative plasmid containing a defined DNA lesion in the 5'-end of the lacZ gene is inserted into a precise locus of E. coli genome reconstituting the entire lacZ gene (Fig 1A). Plasmid integration is irreversible and is achieved by the site-specific recombination system of the bacteriophage lambda, integration events are selected using an antibiotic resistance marker. Using different types of lesioncontaining plasmids, we can directly monitor either TLS (Fig 1B ) or HDGR mechanisms (Fig 1C). For this study, we used two blocking lesions: the UVinduced thymine-thymine pyrimidine (6-4) pyrimidone photoproduct (TT6-4) and the chemical guanine adduct, the N-2-acetylaminofluorene (G-AAF). E. coli possesses three TLS polymerases, involved in the bypass of different types of DNA lesions: Pol II (encoded by polB) [START_REF] Bonner | Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA[END_REF], Pol IV (encoded by dinB) [START_REF] Wagner | The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis[END_REF] and Pol V (encoded by umuDC) [START_REF] Reuven | The mutagenesis protein UmuC is a DNA polymerase activated by UmuD′, RecA, and SSB and is specialized for translesion r e p l i c a t i o n[END_REF]. The expression level of these TLS polymerases is strongly increased upon SOS induction. UV lesions are exclusively bypassed by Pol V while the G-AAF lesion when positioned in a particular sequence context (i.e., the NarI hotspot sequence) can be bypassed either by Pol V (named TLS0 events) or Pol II (named TLS-2 events, frameshift -2) [START_REF] Napolitano | All three SOS-inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis[END_REF][START_REF] Pagès | How DNA lesions are turned into mutations within cells?[END_REF]. The E. coli strains used in this study are deficient for the mismatch repair system (mutS) to prevent corrections of the genetic markers of the integration plasmids, as well as for nucleotide excision repair (uvrA), to avoid excision of the lesion and to focus on lesion tolerance events (see also [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF][START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF]). In our genetic assay, we can insert the lesion either on the lagging or in the leading orientation regarding the replication fork direction. Since we did not observe any significant difference between the two strand orientations, we will present in the manuscript the results obtained for a lesion inserted in the leading strand, while lagging strand results will be presented in Suppl Fig 1.

Loading of RecA: role of the mediators

In a previous study, we have shown that in the presence of a TT6-4 lesion the single mutants recF or recO but also the double mutant recF recO strongly reduced HDGR (Fig 2) [START_REF] Laureti | A non-catalytic role of RecBCD in homology directed gap repair and t r a n s l e s i o n s y n t h e s i s . N u c l e i c A c i d s R e s e a r c h[END_REF]. In the absence of its mediators (i.e. RecF, RecO or RecR), RecA loading can still occur, but randomly and with a slower kinetics [START_REF] Bell | RecA: Regulation and Mechanism of a Molecular Search Engine[END_REF][START_REF] Wu | RecA-SSB interaction modulates RecA nucleoprotein filament formation on SSBwrapped DNA[END_REF]. Since HDGR relies on the recombinase RecA (8), this mechanism is necessarily affected. Similarly, induction of the SOS response is strongly affected by the delay in RecA nucleofilament formation [START_REF] Whitby | Altered SOS induction associated with mutations in recF, recO and recR[END_REF][START_REF] Thoms | Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant[END_REF][START_REF] Hegde | recO and recR mutations delay induction of the SOS response in Escherichia coli[END_REF]. We measured a very low level of Pol V dependent TLS event (<0.5%) because there is essentially no Pol V expression in non-SOS induced cells [START_REF] Sommer | Specific RecA amino acid changes affect RecA-UmuD′ C interaction[END_REF]. In addition, Pol V requires interaction with the tip of a RecA nucleofilament formed by RecFOR complex which less efficiently formed in the recF strain [START_REF] Fujii | RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis[END_REF][START_REF] Raychaudhury | The recombination mediator proteins RecFOR maintain RecA* levels for maximal DNA polymerase V Mut activity[END_REF].

In the presence of a G-AAF lesion, deletion of either recF, recO or recR resulted again in a strong decrease in HDGR events, accompanied this time by a significant increase in Pol II dependent TLS events (Fig 2). The double mutant recF recO showed no further decrease in the level of HDGR compared to the single mutants, neither a significant increase in the level of Pol II dependent TLS (Fig 2). Altogether our genetic data indicate that the recF, recO and recR genes belong to the same epistasis group for lesion bypass and that the three genes play an important role in HDGR, which is expected due to their role in mediating RecA loading.

In a previous study, we have shown that affecting HDGR favors TLS: mutant alleles of RecA, that can form a nucleofilament but are impaired for homology search and strand exchange (i.e. are impaired in the D-loop formation), strongly favor lesion bypass by TLS polymerases [START_REF] Naiman | A defect in homologous recombination leads to increased translesion synthesis in E. coli[END_REF]. However, in this particular context, SOS The graphs represent the parEEon of lesion tolerance pathways (i.e. HDGR, TLS and DCL) for the single mutants recF, recO and recR or the double mutant recF recO in the presence of a TT6-4 lesion* (lea panel) or a G-AAF lesion (right panel). All the strains are compared to our parental strain (WT in the graphs) which is uvrA and mutS deficient. *The data for the TT6-4 lesion have been previously published [START_REF] Laureti | A non-catalytic role of RecBCD in homology directed gap repair and t r a n s l e s i o n s y n t h e s i s . N u c l e i c A c i d s R e s e a r c h[END_REF]. response was constitutively activated thus the amount of TLS polymerases in the cells was significantly increased, which contributed to shifting the balance towards TLS pathway [START_REF] Naiman | A defect in homologous recombination leads to increased translesion synthesis in E. coli[END_REF]. Interestingly, in the recF (as well as recO or recR) deficient strain, despite the absence of SOS induction (SOS response is reduced and strongly delayed in these strains [START_REF] Whitby | Altered SOS induction associated with mutations in recF, recO and recR[END_REF][START_REF] Thoms | Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant[END_REF]), the level of Pol II TLS at a G-AAF lesion increases to a level similar to the one observed after full SOS induction (either by deleting lexA or by UV-induction) (Suppl Fig 2).

Our data point out that TLS can be regulated by factors other than the SOS response: defect in RecA loading is indeed sufficient to strongly increase the level of Pol II dependent TLS events, without activating the SOS response. To further confirm this, we monitored Pol IV activity on a benzo(a)pyrene (BaP): we show that the level of TLS in a recF deficient strain is again increased to the level observed upon full SOS induction by UVirradiation of the cells (Suppl Fig 2).

Effect of the ssDNA gap extension on lesion bypass

We have recently shown that the size of the ssDNA gap can modulate lesion tolerance pathways. Lack of the nuclease RecJ results in a shorter RecA-covered ssDNA gap, thus homology search and strand exchange are less efficient, impacting HDGR mechanism on one hand and favoring TLS pathway on the other hand ( Fig 3 and (33)). While we have previously shown this for the G-AAF lesion, we confirmed that deletion of recJ affects HDGR mechanism also for the TT6-4 lesion (Fig 3).

The helicase RecQ has been proposed to help RecJ in enlarging the ssDNA gap at the 5' end [START_REF] Courcelle | RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli[END_REF]. To confirm this role, we deleted the recQ gene and monitor lesion tolerance pathways in the presence of both G-AAF and TT6-4 lesions. We also observed a decrease in HDGR, but less important than when we deleted recJ or recF (Fig 3). This might indicate that even if the helicase activity of RecQ is required to provide the substrate for the nuclease activity of RecJ, it is not essential or that another helicase, yet to be identified, can partially compensate for the loss of RecQ, as also proposed by Courcelle et al. (32). In the presence of a G-AAF lesion, we measured an increase in Pol II dependent TLS similar to the recJ strain (Fig 3).

Deletion of both recJ and recQ in the presence of either a G-AAF or a TT6-4 lesion showed the same level of lesion tolerance pathways (for both HDGR and TLS) as the single mutant recJ. Hence, from our genetic analyses, recJ and recQ appeared to belong to the same epistasis group for lesion bypass, however RecJ plays a more critical role in HDGR mechanism.

It is interesting to note that in the recJ, recQ and recJ recQ mutants we observe a slight increase in Pol V TLS events (Fig 3 and Suppl Fig 3). In these strains, unlike in the recF deficient strain, RecA loading and nucleofilament formation occurs efficiently allowing the contact with Pol V that is required for its activation [START_REF] Fujii | RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis[END_REF]. This increase is only measured with the G-AAF lesion most likely because the low physiological level of Pol V is a limiting factor in the presence of the strong blocking lesion TT6-4. In line with our findings, Courcelle and coworkers observed in UV irradiated E. coli cells that Pol V was required for survival in the absence of recJ or recQ and that the increase of mutagenesis was Pol V dependent (32, 47).

Altogether, these results further confirm that impairing the extension of the ssDNA gap favors replication by TLS polymerases.

Addi6ve effect of recF and recJ genes

From our data, it appears that both RecA loading and the size of the ssDNA gap are important factors to modulate the partition and the efficiency of lesion tolerance pathways. To further investigate the presynaptic phase, we decided to analyze the genetic interaction between recF and recJ, by deleting both genes and monitoring lesion bypass in the presence of a G-AAF and TT6-4 The graphs represent the parEEon of lesion tolerance pathways (i.e. HDGR, TLS and DCL) for the single mutants recJ* and recQ as well as for the double mutant recJ recQ in the presence of a TT6-4 lesion (lea panel) or a G-AAF lesion (right panel). All the strains are compared to our parental strain (WT in the graphs) which is uvrA and mutS deficient. *The data for recJ in the presence of the G-AAF lesion have been previously published [START_REF] Chrabaszcz | DNA lesions proximity modulates damage tolerance pathways in Escherichia coli[END_REF]. G-AAF lesion. Interestingly, for both lesions the double mutant showed a drastic drop in HDGR events (resulting in less than 10% of HDGR), compensated mainly by damaged chromatid loss (Fig 4). Similar results were obtained for the double mutant recF recQ (Fig 4). These data revealed that recF and recJ (or recQ) genes are not epistatic as it was expected since they are part of the same recombinational RecF pathway [START_REF] Lloyd | Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair[END_REF]. On the contrary, deletion of both genes has an addictive effect on HDGR mechanism. No further effect was instead observed for TLS events: the additional loss in HDGR is compensated by an increase in DCL, not by a further increase in TLS. This is probably due to the fact that in the recF strain, the level of TLS already reaches that of a SOS induced strain (Suppl Fig 2 ) and cannot increase further. Likewise, no significant increase in Pol IV dependent TLS was observed when we deleted both recF and recJ genes in the presence of a BaP lesion (Suppl Fig 2). A similar phenotype, i.e. a strong loss of HDGR, was previously observed in a recA deficient strain (see Fig 4 and also [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF]). However, in the absence of RecA, we do not observe an increase in TLS. This might be explained by the fact that in the absence of RecF, RecA loading can still occur (but less efficiently) and this is sufficient to help stabilizing the ssDNA gap favoring replication by Pol II TLS. Similarly, a better stability of the ssDNA gap could explain why we did not observe a drastic decrease in cell survival for the double mutant recF recJ as in a recA deficient strain. To note, the 10% of sectored blue-white colonies still observed in the recF recJ (and recF recQ) strain as well as in the recA deficient strain result from a still uncharacterized RecAindependent mechanism, as previously proposed by the Rosenberg laboratory [START_REF] Xia | Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase[END_REF][START_REF] Lovett | Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecAindependent pathways[END_REF].

Altogether these results indicate that both the loading of RecA and the size of the ssDNA gap are equally important for an efficient HDGR. Indeed, the simultaneous deficiency of these two processes condemns cells to survive mostly on damaged chromatid loss.

DISCUSSION

In this study we investigated the role of the presynaptic phase of the RecF pathway on lesion bypass. The presynaptic phase is involved in the processing of the ssDNA gap formed upon encountering a lesion, and ends with the formation of a RecA nucleofilament. Once formed, the nucleofilament will engage in homology search and strand pairing, resulting in a D-loop structure that promotes HDGR and prevents TLS. The use of our single lesion assay allows to finely explore what occurs in the presence of a single replication blocking lesion and to better decipher lesion bypass mechanisms.

The presynaptic phase of the RecF pathway comprises several proteins that were expected to belong to the same epistasis group [START_REF] Lloyd | Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair[END_REF]. While we confirmed this for the recF-recO-recR genes as well as for recQ-recJ, we unveiled an additive effect between recF and recJ (and recF-recQ), that results in a drastic loss of HDGR mechanism reminiscent of a recA deficient phenotype. We also showed that a defect in RecA loading and nucleofilament formation, as in a recF strain, or a defect in ssDNA gap extension, as in recJ strain, not only strongly affects HDGR mechanism but also promotes replication by Pol II or Pol IV TLS in the absence of SOS induction (i.e. without increasing the amount of TLS polymerases).

In non-stressed conditions, tolerance of highly blocking lesions relies almost exclusively on HDGR while TLS is a minor pathway [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF]. We have already shown that several factors contribute to regulate the balance between HDGR and TLS. In bacteria, TLS is positively regulated by the SOS response that increases the amount of the TLS polymerases in the cell, but TLS can also be modulated by HDGR efficiency: RecA mutants impaired in D-loop formation show a defect in HDGR and an increase in TLS [START_REF] Naiman | A defect in homologous recombination leads to increased translesion synthesis in E. coli[END_REF]. Similarly, the The graphs represent the parEEon of lesion tolerance pathways (i.e. HDGR, TLS and DCL) for the double mutant recF recJ and recF recQ in the presence of a TT6-4 lesion (lea panel) or a G-AAF lesion (right panel). All the strains are compared to our parental strain (WT in the graphs) which is uvrA and mutS deficient. The data for the recA deficient strain have been already published [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF].

proximity of lesions on opposite strands generates overlapping ssDNA regions that prevent HDGR, leading again to an increase in TLS [START_REF] Chrabaszcz | DNA lesions proximity modulates damage tolerance pathways in Escherichia coli[END_REF]. In this work, we show that it is specifically the presynaptic phase of the RecF pathway that is critical for lesion tolerance pathway choice. Indeed, impairing the presynaptic phase leads to a delay in the formation of an efficient D-loop and therefore increases the time window allowed for TLS to occur, leading to a significant increase in TLS.

Based on these data, we propose a model that illustrates how the choice of lesion tolerance pathways is directed during the presynaptic phase of the RecF pathway (Fig 5). In the presence of an unrepaired lesion, the replicative polymerase temporarily stalls before a repriming event allows replication to continue, leaving a ssDNA gap. SSB protein will immediately cover the ssDNA gap to protect it and to orchestrate the recruitment of several proteins. SSB was indeed shown to directly interact with most of the proteins of the presynaptic phase as well as with the TLS polymerases [START_REF] Shereda | SSB as an organizer/mobilizer of genome maintenance complexes[END_REF][START_REF] Bianco | The tale of SSB[END_REF]. The SSB-coated ssDNA gap can be regarded as a platform for protein recruitment for the processing of the ssDNA gap and for lesion bypass. The 5' ds-ssDNA junction of the ssDNA gap appears to be a shared substrate for both RecQJ and RecFR complexes. Whether and which factors During replicaEon, unrepaired lesions can temporarily block the replicaEve polymerase (a). The polymerase can skip the lesion and restart replicaEon downstream, leaving a ssDNA gap (b). SSB protein is immediately recruited to the ssDNA gap (c) to protect it from undesired degradaEon, but also to orchestrate the recruitment of the proteins involved in the processing and the filling in of the ssDNA gap. The helicase RecQ and the nuclease RecJ are loaded at the 5' ds-ssDNA juncEon and their concerted acEon enlarge the size of the gap (d). The processivity of RecJ-RecQ drops and they are replaced by the RecFR complex that is also recruited at the 5' juncEon (e). The acEon of RecFR and of the RecOR complex at the ssDNA gap is to displace SSB and load the regulate the timing of RecQJ and RecFR recruitment is not known yet, however, we suggest that RecQ and RecJ are the first to be recruited at the 5' junction of the nascent DNA. We based this hypothesis on the fact that RecQ and RecJ were shown to directly interact with SSB in the presence of a ssDNA gap and this interaction stimulates their catalytic activity to enlarge the ssDNA gap [START_REF] Morimatsu | RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination[END_REF][START_REF] Cheng | Structural basis for DNA 5-end resection by RecJ[END_REF][START_REF] Han | RecJ exonuclease: substrates, products and i n t e r a c t i o n w i t h S S B . N u c l e i c A c i d s R e s e a r c h[END_REF][START_REF] Shereda | A central role for SSB in Escherichia coli RecQ DNA helicase function[END_REF]. Supporting our hypothesis, Xia and coworkers [START_REF] Xia | Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase[END_REF] showed that RecJ and RecQ promote recombination and Holiday junction formation in vegetative E. coli cells, acting upstream of RecA during the repair of ssDNA gaps. Therefore, the extension of the gap must come before the loading of RecA. We have previously estimated the size of the ssDNA gaps to be in the range of 1.8-3.5 Kb (33) which correlates with the processivity established in vitro for RecJ [START_REF] Han | RecJ exonuclease: substrates, products and i n t e r a c t i o n w i t h S S B . N u c l e i c A c i d s R e s e a r c h[END_REF]. Once the complex RecQJ falls off, RecFR complex can have access to the 5' ds-ssDNA junction and this also allows the recruitment of the RecOR complex to the ssDNA gap where RecO directly interacts with SSB [START_REF] Umezu | Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by singlestranded DNA-binding protein[END_REF][START_REF] Ryzhikov | Mechanism of RecO recruitment to DNA by singlestranded DNA binding protein[END_REF]. Finally, RecA loading and filament formation can start. Since it occurs preferentially in the direction 5'->3' [START_REF] Bianco | RecA Protein -eLS -Bianco -Wiley Online Library[END_REF], it will inhibit a new RecQ-RecJ recruitment. Once the RecA nucleofilament on the ssDNA gap is formed it will engage in homology search and strand invasion, forming the so-called D-loop structure required for the HDGR mechanism, that we showed to be the major lesion tolerance pathway employed by cells under non-stressed conditions [START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF][START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF].

Our data show that defect in the genes of the presynaptic phase affects HDGR mechanism favoring TLS pathway. Indeed, in the absence of either RecJ or RecQ, the size of ssDNA gap in not enlarged, thus impairing the efficiency of HDGR mechanism. Likewise, deletion of the RecA mediators (recF, recO or recR) causes a significant delay in RecA nucleofilament formation, as shown in vitro where nucleation time goes from 2-10 min in the presence of RecFOR to 10-60 min in its absence [START_REF] Bell | RecA: Regulation and Mechanism of a Molecular Search Engine[END_REF]. In both cases, we observed an increase in TLS pathways. Absence of RecF, other than causing a delay in RecA filament formation, could allow RecJ to be recruited again and further extend the gap, a too long ssDNA gap can also affect HDGR mechanism. Indeed, in recF strain it was observed a RecJ dependent degradation of the nascent DNA that caused a delay in replication resumption [START_REF] Courcelle | recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli[END_REF][START_REF] Chow | RecO acts with RecF and RecR to protect and maintain replication forks blocked by UVinduced DNA damage in Escherichia coli[END_REF]. Finally, the concomitant deficiency of RecF and RecJ results in a drastic decrease of HDGR mechanism, indicating that the slow formation of a small RecA-covered ssDNA strongly inhibits HDGR.

From our model, we can evince that the presynaptic phase, that includes the different steps t o a c h i e v e t h e f o r m a t i o n o f t h e R e c A nucleofilament, is crucial in modulating lesion tolerance pathways and in turn the outcome of lesion bypass (non-mutagenic versus mutagenic). The presynaptic phase can be considered as the "time window" where TLS can take place. Once the D-loop is formed, the recombinogenic RecA filament engages in HDGR process, thus closing the window that was allowed for TLS. Any delay in the presynaptic phase will extend this time window leading to an increase in TLS.

Figure 1 :

 1 Figure 1: Lesion tolerance assay and lesion containing plasmids. A)The system is based on the phage lambda site-specific recombinaEon and it requires a recipient E. coli strain with a single a)R site fused to the 3'-end of the lacZ gene and a non-replicaEng plasmid construct containing a single DNA lesion (red triangle) in the 5'-end of the lacZ gene fused to the a)L site. The recombinaEon reacEon between a)L and a)R is controlled by ectopic expression of phage lambda int-xis proteins (provided by pVP135), and leads to the integraEon of the lesion-containing vector into the chromosome. Integrants are selected on the basis of their resistance to ampicillin and the chromosomal integraEon restores an enEre lacZ gene. B) Plasmid duplexes used to monitor TLS events (see also[START_REF] Pagès | Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome[END_REF]. The non-damaged strand (A+B markers) contains a short sequence heterology opposite the lesion that inacEvates the lacZ gene and serves as a geneEc marker for strand discriminaEon. Only the replicaEon by TLS polymerases of the A+C markers will give rise to a funcEonal lacZ gene and therefore to a sectored blue-white colony. For the G-AAF lesion, one construct measures TLS0 events specific of Pol V-dependent TLS events, while another one contains a +2 frameshit that allows to measure TLS-2 events specific of Pol II when the G-AAF lesion is inserted in the NarI hotspot sequence (see[START_REF] Pagès | How DNA lesions are turned into mutations within cells?[END_REF]. C) Plasmid duplexes used to specifically monitor strand exchange mechanisms, i.e. HDGR events (see alsoLaureE et al., 2015). The system is a modified version of the plasmid constructs used to monitor TLS events. In these plasmids four geneEc markers have been designed in order to disEnguish the replicaEon of the non-damaged strand (containing markers A and B) from the damaged strand (containing markers C and D). Using a combinaEon of frameshia and stop codon, we inacEvated lacZ gene on both the damaged (C-D) and undamaged (A-B) strands of the vector. Only a strand exchange mechanism by which replicaEon has been iniEated on the damaged strand (incorporaEon of marker C), and where a template switch occurred at the lesion site (leading to incorporaEon of marker B) will restore a funcEonal lacZ gene (the combinaEon of markers C and B contains neither a stop codon nor a frameshia); therefore leading to sectored bluewhite colonies. When the damaged strand is loss, only the A and B marker will be replicaEng giving rise to a white colony.

(

  : lacZ-C + B: lacZ+ Homology Directed Gap Repair pLL1/2c for TT6-4 lesion pLL1/7 for G-AAF lesion Pagès et al., 2012) (Laureti et al., 2015) A B. Plasmid constructions to monitor TLS: C. Plasmid constructions to monitor HDGR: pVP141/142: G-AAF lesion TLS0 (Pol V) pVP143/144: G-AAF lesion TLS-2 (Pol II) pGP1/2: TT6-4 lesion (Pol V) pVP143/pGP9: BaP lesion (Pol IV)

5

 5

Figure 2 .

 2 Figure 2. Deficiency in RecA loading promotes replica6on through TLS polymerases.The graphs represent the parEEon of lesion tolerance pathways (i.e. HDGR, TLS and DCL) for the single mutants recF, recO and recR or the double mutant recF recO in the presence of a TT6-4 lesion* (lea panel) or a G-AAF lesion (right panel). All the strains are compared to our parental strain (WT in the graphs) which is uvrA and mutS deficient. *The data for the TT6-4 lesion have been previously published[START_REF] Laureti | A non-catalytic role of RecBCD in homology directed gap repair and t r a n s l e s i o n s y n t h e s i s . N u c l e i c A c i d s R e s e a r c h[END_REF].

6

 6

Figure 3 .

 3 Figure 3. The size of the ssDNA gap affects the efficiency of HDGR mechanism favouring TLS.The graphs represent the parEEon of lesion tolerance pathways (i.e. HDGR, TLS and DCL) for the single mutants recJ* and recQ as well as for the double mutant recJ recQ in the presence of a TT6-4 lesion (lea panel) or a G-AAF lesion (right panel). All the strains are compared to our parental strain (WT in the graphs) which is uvrA and mutS deficient. *The data for recJ in the presence of the G-AAF lesion have been previously published[START_REF] Chrabaszcz | DNA lesions proximity modulates damage tolerance pathways in Escherichia coli[END_REF].

Figure 4 .

 4 Figure 4. Addi6ve effect of recF and recJ results in loss of HDGR mechanism.The graphs represent the parEEon of lesion tolerance pathways (i.e. HDGR, TLS and DCL) for the double mutant recF recJ and recF recQ in the presence of a TT6-4 lesion (lea panel) or a G-AAF lesion (right panel). All the strains are compared to our parental strain (WT in the graphs) which is uvrA and mutS deficient. The data for the recA deficient strain have been already published[START_REF] Laureti | Bacterial proliferation: keep dividing and don't mind the gap[END_REF].

Figure 5 .

 5 Figure 5. The presynap6c phase of the RecF pathwayHere is represented a scheme of the presynapEc phase during lesion bypass in the presence of a replicaEon-blocking lesion (red triangle) in the leading strand. For simplicity aaer the repriming event (b), we have drawn only the molecular mechanisms occurring on the ssDNA gap. During replicaEon, unrepaired lesions can temporarily block the replicaEve polymerase (a). The polymerase can skip the lesion and restart replicaEon downstream, leaving a ssDNA gap (b). SSB protein is immediately recruited to the ssDNA gap (c) to protect it from undesired degradaEon, but also to orchestrate the recruitment of the proteins involved in the processing and the filling in of the ssDNA gap. The helicase RecQ and the nuclease RecJ are loaded at the 5' ds-ssDNA juncEon and their concerted acEon enlarge the size of the gap (d). The processivity of RecJ-RecQ drops and they are replaced by the RecFR complex that is also recruited at the 5' juncEon (e). The acEon of RecFR and of the RecOR complex at the ssDNA gap is to displace SSB and load the

ACKNOWLEDGEMENT

We thank Jean-Hugues Guervilly and Florencia Villafañez for critical reading of the manuscript.

FUNDING

This work was supported by the Agence Nationale de la Recherche (ANR) Grant [GenoBlock ANR-14-CE09-0010-01]; and the Fondation pour l a R e c h e r c h e M é d i c a l e E q u i p e F R M -EQU201903007797.