Shingo Fujii
email: shingo.fujii@inserm.fr

Robert P Fuchs
email: robert.fuchs@inserm.fr

A Comprehensive View of Translesion Synthesis in Escherichia coli and beyond

The RecA-Fluttering Model How is Pol V activated on RecA-coated ssDNA

INTRODUCTION WHAT HAPPENS WHEN A REPLICATION FORK ENCOUNTERS A LESION? CHARACTERISTIC FEATURES OF DNA POLYMERASES IN E. COLI

What Is the Mechanism Underlying Functional Access of Pol IV on Template DNA?

The Pivotal Role of RecA for Assembly of Functional Pol V Characteristic Features of Pol IV-and Pol V-Mediated TLS Pathways Translesion synthesis, gap filling synthesis, post-replicative gap repair, untargeted mutagenesis, replicative polymerase, Pol IV, Pol V, RecA, β-clamp, adaptive mutation, evolution

INTRODUCTION

Cells have evolved robust and versatile defense mechanisms to cope with various types of DNA damages derived from endogenous and exogenous sources. Endogenous DNA damage stems from normal metabolic processes (e.g., reactive oxygen species (ROS) via respiration of mitochondria (1)) or intrinsic instability of DNA per se (e.g., abasic lesion (apurinic/apyrimidinic (AP) site) [START_REF] Lindahl | Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid[END_REF]). The steady-state number of DNA damages caused by endogenous sources is estimated to be over 40,000 in human DNA per cell (AP site is the most prevalent damage and is estimated to be ≈30,000) [START_REF] Nakamura | The endogenous exposome[END_REF]. With respect to exogeneous sources, they are derived from numerous chemicals (e.g., polycyclic aromatic hydrocarbons (PAHs) formed during incomplete combustion in cigarette smoke or car exhausts (4); alkylating agents during chemotherapy [START_REF] Cheung-Ong | DNA-Damaging Agents in Cancer Chemotherapy: Serendipity and Chemical Biology[END_REF]) or environmental sources (e.g., UV light via sunshine [START_REF] Leclerc | The thymine-thymine pyrimidinepyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3' thymine-to-cytosine transitions in Escherichia coli[END_REF]). Most DNA damage is effectively repaired through canonical DNA repair pathways such as nucleotide excision repair (NER) and base excision repair (BER) [START_REF] Friedberg | DNA Repair and Mutagenesis[END_REF]. If impaired in such repair pathways, cells become highly sensitive to DNA damages [START_REF] Courcelle | RecA-Dependent Recovery of Arrested DNA Replication Forks[END_REF]. However, even in the presence of efficient DNA repair systems, a subfraction of DNA damage escapes repair and has the opportunity to encounter the replicative DNA polymerase (Pol) at the fork (step a in Fig. 1). In case of replication blocking lesions, replication forks skip over the DNA damage and re-initiate downstream leaving a single-stranded (ss) DNA gap [START_REF] Lehmann | Gaps and forks in DNA replication: Rediscovering old models[END_REF]. As a consequence, the unrepaired lesion is now located in the context of ssDNA (step b in Fig. 1) where it becomes irreparable by regular DNA repair systems that only function on double-stranded (ds) DNA. In order to cope with DNA damage located in ssDNA, cells have evolved two so-called DNA damage tolerance strategies that take place in the context of postreplicative gap repair (Fig. 1). The most straightforward damage tolerance strategy involves specialized DNA Pols that bypass directly DNA lesions, a process called translesion synthesis (TLS) (step c in Fig. 1) [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF][START_REF] Marians | Lesion Bypass and the Reactivation of Stalled Replication Forks[END_REF][START_REF] Henrikus | Specialised DNA polymerases in Escherichia coli: roles within multiple pathways[END_REF]. Whereas some types of DNA lesions are bypassed by the replicative Pols themselves (e.g., 8-oxo-dG) [START_REF] Hsu | Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase[END_REF], in this review we define TLS as an event requiring the action of a specialized Pol (i.e., TLS Pol). TLS is potentially highly mutagenic when DNA lesions are non-instructive (e.g., AP site; TT pyrimidine-pyrimidone (6-4) photoproduct (TT ). Therefore, the resulting nascent strand often contains a mutation opposite the template lesion site (a process referred to as "targeted mutagenesis").

The second DNA damage tolerance strategy, referred to as homology-dependent gap repair (HDGR) or homology-directed repair (HDR) operated through template switching (for reviews see [START_REF] Marians | Lesion Bypass and the Reactivation of Stalled Replication Forks[END_REF][START_REF] Fujii | Chronological Switch from Translesion Synthesis to Homology-Dependent Gap Repair In Vivo[END_REF]), aims at relocating the lesion into a double-stranded context by means of a homologous recombination intermediate with the complementary sister chromatid (step f in Fig. 1) [START_REF] Courcelle | RecA-Dependent Recovery of Arrested DNA Replication Forks[END_REF][START_REF] Marians | Lesion Bypass and the Reactivation of Stalled Replication Forks[END_REF][START_REF] Bichara | RecA-mediated excision repair: a novel mechanism for repairing DNA lesions at sites of arrested DNA synthesis[END_REF][START_REF] Bichara | Preferential postreplication repair of DNA lesions situated on the leading strand of plasmids in Escherichia coli[END_REF][START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF]. Irrespective of the precise model, the main purpose of HDGR (that is also the case for TLS) is to prevent deleterious discontinuities in DNA strands and to reinstate the DNA damage from its irreparable ssDNA context to a repairable dsDNA context (steps c-e or f-h in Fig. 1). Let us note that a similar template switching mechanism, usually referred to as fork regression or fork reversal, has been proposed to allow a replication fork, arrested in the vicinity of a lesion or a secondary structure in the leading strand template, to restart [START_REF] Marians | Lesion Bypass and the Reactivation of Stalled Replication Forks[END_REF][START_REF] Michel | Replication Fork Breakage and Restart in Escherichia coli[END_REF].

Owing to the intrinsic low-fidelity of TLS Pols, the process of TLS is highly mutagenic both at the lesion bypass step per se (targeted mutagenesis) and downstream from the lesion on normal template DNA (untargeted mutagenesis) (steps b to c in Fig. 1).

As discussed later in this review, despite their high mutagenic load, TLS pathways are present in most species [START_REF] Ohmori | The Y-family of DNA polymerases[END_REF][START_REF] Goodman | Error-Prone Repair DNA Polymerases in Prokaryotes and Eukaryotes[END_REF] strongly suggesting that TLS pathways are likely to be beneficial for evolution in the long term and for prevention of persistent ssDNA gaps in the short term, rather than being an extra back-up pathway.

The bypass of many different DNA lesions by different TLS Pols has been extensively studied in vitro [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF][START_REF] Zhao | Translesion Synthesis: Insights into the Selection and Switching of DNA Polymerases[END_REF]. However, the overall process of TLS in vivo has received little attention until recently [START_REF] Kochenova | DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNA[END_REF][START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. The present article aims at discussing how the various Pols that are encoded and expressed at different levels in E. coli manage the overall process of post-replicative gap repair in vivo. We will propose a TLS model involving an interplay between the error-prone TLS Pols and the high-fidelity Pols in the context of physiologically relevant conditions, and discuss its biological consequences.

WHAT HAPPENS WHEN A REPLICATION FORK ENCOUNTERS A LESION?

When an ongoing replication fork encounters a replication blocking lesion in one of the template strands, it was initially expected that the fate of the fork would depend on whether the lesion is located in the leading or the lagging strand template. For a lesion in the lagging strand, the fork would go ahead leaving a gap (i.e., an unfinished Okazaki fragment). With a leading strand lesion, the fork may stall due to the concept of continuous leading strand replication as observed with reconstituted replication forks in vitro [START_REF] Wu | Coordinated leading-and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size[END_REF][START_REF] Kornberg | DNA Replication[END_REF].

However, initial data from Okazaki indicated that, in E. coli, normal replication is discontinuous in both leading and lagging strands [START_REF] Okazaki | Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains[END_REF]. Discontinuous strand synthesis was also demonstrated when E. coli is damaged by UV irradiation [START_REF] Rupp | Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation[END_REF] suggesting that the fork can skip over UV-induced lesions during both leading and lagging strand synthesis.

The reason for which such observations had not been taken into account came from the lack of biochemical evidence for leading strand repriming. Nearly 40 years after the classic in vivo observations, it was shown that repriming events relying on DnaG primase can occur during leading strand synthesis in vitro, either in the presence of accessory factors related to de novo priming activities (PriA-or PriC-dependent pathway) [START_REF] Heller | Replication fork reactivation downstream of a blocked nascent leading strand[END_REF] or in the absence of such factors [START_REF] Yeeles | The Escherichia coli replisome is inherently DNA damage tolerant[END_REF]. In addition, an alternative pathway for skipping over a lesion on leading strand was also proposed based on in vitro observation that a leading strand polymerase can utilize a mRNA formed downstream of the leading strand template DNA [START_REF] Pomerantz | The replisome uses mRNA as a primer after colliding with RNA polymerase[END_REF]. These observations led to the suggestion that repriming of leading strand synthesis can occur downstream from a replication-blocking lesion leaving a gap behind the fork. As a consequence, the process of TLS became regarded as part of postreplicative gap repair for both leading and lagging strand lesions, rather than a continuous process occurring at the fork [START_REF] Lehmann | Gaps and forks in DNA replication: Rediscovering old models[END_REF], sometimes referred to as "TLS on the fly" [START_REF] Hedglin | Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle[END_REF].

Discontinuous DNA replication of both leading and lagging strands was also supported in vivo using a ligase mutant [START_REF] Amado | The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision[END_REF][START_REF] Amado | Low-molecular-weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity[END_REF] and as well as by fluorescence-based imaging in live E. coli cells as discussed below [START_REF] Beattie | Frequent exchange of the DNA polymerase during bacterial chromosome replication[END_REF]. Indeed, the scenario of TLS being implemented behind the replication fork, was supported via single molecule fluorescence assays in which the localization of a TLS Pol, Pol V, was shown to be distinct from the localization of the Pol III replisome following UV irradiation in vivo [START_REF] Robinson | Regulation of Mutagenic DNA Polymerase V Activation in Space and Time[END_REF]. Similarly, single molecule fluorescence assays in vivo also indicated that Pol IV, another TLS Pol, principally colocalizes outside of the replisome following exposure of DNA damaging agents (e.g., 4-nitroquinoline N-oxide (4-NQO)), implying that Pol IV-mediated TLS also predominantly occurs behind rather than at the replication fork [START_REF] Henrikus | DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli[END_REF].

CHARACTERISTIC FEATURES OF DNA POLYMERASES IN E. COLI

E. coli possesses five DNA polymerases (Pol I to V). Whereas classical Pols (i.e., Pol I to III) were shown to exhibit bypass properties for some lesions under certain experimental conditions [START_REF] Paz-Elizur | Mechanism of translesion DNA synthesis by DNA polymerase II. Comparison to DNA polymerases I and III core[END_REF][START_REF] Paz-Elizur | Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I[END_REF][START_REF] Nevin | Replisome-mediated translesion synthesis by a cellular replicase[END_REF], their contribution to TLS under physiological conditions, is limited (largely unknown) except for a few examples [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF]. In contrast, the two remaining Pols (i.e., Pol IV and V) have characteristic features of TLS Pols, namely their lack of proofreading function and their capacity to accommodate bulky lesions given their spacious catalytic domain [START_REF] Ohmori | The Y-family of DNA polymerases[END_REF][START_REF] Lee | Homology modeling of four Y-family, lesion-bypass DNA polymerases: The case that E. coli Pol IV and human Pol κ are orthologs, and E. coli Pol V and human Pol η are orthologs[END_REF]. Interestingly, Pol IV and Pol V appear to be adapted for the bypass of minor and major groove lesions (i.e., division of labor), respectively [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF].

It may appear awkward to refer to minor versus major groove location during TLS as the lesion is located at the ss to dsDNA junction (step b in Fig. 1). However, in the Pol active site, the conformation of the damaged nucleotide (at the ss to dsDNA junction) may adopt, during the nucleotide insertion step, the same conformation as in dsDNA, allowing thus for Pol IV versus Pol V selectivity. Of note, TLS polymerase discrimination at the insertion step is undoubtedly performed through stochastic accesses of Pols (trial and error) [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF], rather than during a scenario where a lesion would specifically recruit a Pol that is best suited for its bypass.

When a replicative Pol stalls at a replication blocking lesion and then dissociates, a 3'-end of the nascent strand is typically located one nucleotide before the lesion (42); upon dissociation, the stalled Pol leaves its processivity factor (the β-clamp) bound to the template DNA [START_REF] Fujii | Defining the position of the switches between replicative and bypass DNA polymerases[END_REF][START_REF] Fujii | Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways[END_REF]. Downstream of the lesion, an exposed ssDNA gap becomes first coated by ssDNA binding (SSB) proteins that are subsequently replaced by a RecA filament [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF][START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF][START_REF] Fujii | RecFOR proteins are essential for Pol Vmediated translesion synthesis and mutagenesis[END_REF][START_REF] Bell | Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA[END_REF] forming the so-called SOS signal, the bacterial stress response [START_REF] Friedman | Precise temporal modulation in the response of the SOS DNA repair network in individual bacteria[END_REF][START_REF] Michel | After 30 years of study, the bacterial SOS response still surprises us[END_REF][START_REF] Culyba | Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network[END_REF]. Any Pols (or other types of DNA binding proteins like exonucleases and helicases [START_REF] Courcelle | RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli[END_REF]) can stochastically access the vacant 3'-end of the nascent strand. If a classical Pol (i.e., Pol I to III) accesses the 3'-end in the vicinity of a lesion (e.g., TT (6-4)), it either quickly detaches as a consequence of non-productive association/dissociation cycles (Fig. 2A) or causes degradation/resynthesis cycles upstream from the lesion site [START_REF] Fujii | Defining the position of the switches between replicative and bypass DNA polymerases[END_REF][START_REF] Fujii | Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways[END_REF].

What Is the Mechanism Underlying Functional Access of Pol IV on Template DNA?

If a lesion is located in the minor groove (e.g., [-ta]-benzo[a]pyrene-N 2 -dG ([-ta]-BaP-dG)), Pol IV bypasses the lesion in vivo [START_REF] Seo | Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli[END_REF]. Pol IV first accesses a 3'-end of nascent strand in the vicinity of the lesion and subsequently interacts with a freely sliding β-clamp, leading to the formation of a functional complex (Pol IV-fc) (Fig. 2B). It should be stressed that the opposite scenario (i.e., the β-clamp recruits Pols) is unlikely since the β-clamp is freely sliding on DNA [START_REF] Kong | Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp[END_REF]. Interaction with the β-clamp is genetically essential for Pol IVmediated TLS (52); moreover, it was shown that, under multiple-hit conditions in vitro, the processivity of Pol IV is only one nucleotide in the absence of the β-clamp [START_REF] Wagner | The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis[END_REF][START_REF] Wagner | The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity[END_REF], indicating high instability of Pol IV on T/P. These observations imply that only a minor fraction of transiently bound Pol IV will succeed to interact with a freely sliding β-clamp. In conclusion, the rate limiting factor for successful TLS by Pol IV is the formation of Pol IVfc (Fig. 2B).

Intriguingly, under normal growth conditions, the basal expression level of Pol IV (≈250 molecules per cell), is the second most abundant among all Pols and is further induced to ≈2,500 molecules per cell under SOS induction [START_REF] Kim | Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli[END_REF]. One may conclude that the high abundance of Pol IV counteracts its intrinsically low T/P binding affinity by increasing the probability of stochastic access thus allowing the formation of Pol IV-fc required for the minor groove adduct bypass. This notion is supported by the observation that Pol IV-mediated TLS across [-ta]-BaP-dG adducts in vivo indeed occurs under non-SOS induced condition and is only moderately stimulated by SOS induction (S. Fujii, A.

Isogawa, R. Fuchs, unpublished data). In contrast, on normal T/P, it is known that Pol IVinduced untargeted mutagenesis in vivo was below detection limits even under SOS induced conditions, while being only detected upon artificially high overexpression of Pol IV [START_REF] Kim | Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA[END_REF][START_REF] Slechta | Adaptive mutation: general mutagenesis is not a programmed response to stress but results from rare coamplification of dinB with lac[END_REF]. These observations suggest that the number of Pol IV molecules required for stochastic access during TLS is below the number required for access on normal T/P.

Under physiological levels of expression, Pol IV cannot efficiently compete with classical

Pols on normal T/P given its instability in the absence of interaction with the β-clamp [START_REF] Wagner | The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity[END_REF].

In contrast, when a post-replicative ssDNA gap contains a minor groove lesion, owing to the lack of elongation capacity by classical Pols, multiple accesses of Pol IV can lead to formation of a functional complex (Pol IV-fc) by interaction with a freely sliding β-clamp.

Formation of this functional complex is thus restricted to Pol IV specific substrates such as minor groove lesions.

The Pivotal Role of RecA for Assembly of Functional Pol V

If a lesion is located in the major groove (e.g., N-2-acetylaminofluorene-dG (AAF-dG) (58)), Pol V bypasses the lesion in vivo [START_REF] Napolitano | All three SOSinducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis[END_REF]. For Pol V-mediated TLS, interaction with RecA is essential as demonstrated both genetically (60-62) and biochemically [START_REF] Reuven | The mutagenesis protein UmuC is a DNA polymerase activated by UmuD', RecA, and SSB and is specialized for translesion replication[END_REF][START_REF] Tang | UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V[END_REF].

Interaction of Pol V with the β-clamp is also genetically essential for Pol V-mediated TLS [START_REF] Becherel | Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E. coli cells[END_REF], as previously mentioned for Pol IV. Subsequently, both genetic requirements (i.e., interaction with both the β-clamp and RecA) were also demonstrated to be essential factors in vitro [START_REF] Fujii | The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited[END_REF]. When Pol V accesses a lesion-containing T/P in the vicinity of a RecA-coated ssDNA gap, the following scenario is likely to occur (Fig. 2C). The interaction of Pol V with T/P is, as already noted for Pol IV, highly unstable [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]; the RecA molecule located at a 3'-tip of the RecA nucleoprotein filament (RecA*) stabilizes a Pol V molecule transiently bound to the T/P, leading to the formation of a complex referred to as pre-Pol V-fa (pre-Pol V functional assembly: Pol V with RecA*). The resulting increase in residency time allows Pol V to encounter and to associate with a freely sliding β-clamp. Consequently, a functional assembly of Pol V with RecA* and the β-clamp (Pol V-fa) is reached, allowing TLS to ensue (Fig. 2C) [START_REF] Fujii | The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited[END_REF][START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF].

A single RecA molecule in RecA* typically occupies 3 nt [START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF]; the length of the ssDNA gap between a 3'-end of primer and a 3'-tip of RecA* is therefore 0, 1 or 2 (modulo 3) nt long. Potentially, only one of the three gaps has the proper size to form a pre-Pol Vfa. To allow formation of a correct pre-Pol V-fa complex, the adjustment of the gap size may occur owing to the intrinsic dynamic features of RecA* (67), or as a result of the dynamic state of 3'-end of primer that is subject to degradation/resynthesis cycles by classical Pols as mentioned above [START_REF] Fujii | Defining the position of the switches between replicative and bypass DNA polymerases[END_REF].

Characteristic Features of Pol IV-and Pol V-Mediated TLS Pathways

The major difference between Pol IV and Pol V in terms of their respective activation procedure for TLS is the unique requirement of RecA for Pol V. Under SOSinduced conditions, the estimated number of Pol V (15 ~ 60 molecules per cell) ( 68) is ≈40 ~ 170-fold lower than that of Pol IV (~2,500 molecules per cell) [START_REF] Kim | Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli[END_REF]. Pol IV and Pol V implement different strategies to compensate for their low T/P affinity. For Pol IV, its high abundance compensates for its low affinity to T/P, while Pol V qualitatively benefits from additional stabilization through a specific interaction with RecA. During SOS induction, in contrast to dinB encoding Pol IV that is induced early (69), the umuDC genes are induced late [START_REF] Crowley | Answering the Call: Coping with DNA Damage at the Most Inopportune Time[END_REF] and the UmuD'2C (i.e., Pol V) appears only ≈50 minutes after SOS induction [START_REF] Pham | Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli[END_REF][START_REF] Rangarajan | A phenotype for enigmatic DNA polymerase II: a pivotal role for pol II in replication restart in UV-irradiated Escherichia coli[END_REF]. In case of a major groove lesion like AAF-dG, given that Pol V induction is delayed to a late time point during SOS induction, one can assume that all other Pols have randomly accessed the 3'-end of primer in a non-productive way (Fig. 2A). Thus, depending upon the type of lesion to be bypassed (i.e., minor or major groove lesion), the timing of TLS occurrence largely differs between Pol IV-and Pol V-mediated pathways. Since bypass across [-ta]-BaP-dG in vivo does not require SOS induction, Pol IV-mediated TLS can occur at an earlier time compared to TLS mediated by Pol V, reducing thus the time harmful ssDNA is exposed. This notion is also supported by single molecule fluorescence assays in vivo [START_REF] Henrikus | DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli[END_REF].

UNTARGETED MUTAGENESIS DURING COMPLETION OF THE WHOLE PROCESS OF POL V-MEDIATED TLS

In case cells would aim to minimize mutagenesis during lesion bypass (Fig. 3A), one would assume that the error-prone TLS Pols would dissociate from the template DNA as soon as a TLS patch reaches a minimal length (pink line in Fig. 1c) sufficient for extension by high-fidelity Pols (e.g., Pol III) [START_REF] Fujii | Defining the position of the switches between replicative and bypass DNA polymerases[END_REF].

We have previously shown that a 5 nt long TLS patch is efficiently extended by Pol III [START_REF] Fujii | Defining the position of the switches between replicative and bypass DNA polymerases[END_REF]. In such a case, the occurrence of untargeted mutations would be exclusively limited to a short TLS patch (Fig. 3B). However, for Pol V-mediated TLS in vivo, unexpectedly, untargeted mutagenesis is spread over essentially the whole postreplicative gap and even in the upstream region (Fig. 3C) [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. Our work revealed that the error frequency of Pol V-induced untargeted mutagenesis during the gap filling synthesis lies in the 10 -5 range per base. If a gap size is 1 kb, then cumulative untargeted mutations reach a 10 -2 frequency for every TLS event. The data clearly indicate that, in vivo, access of Pol V to undamaged template DNA is not strictly limited to a minimal TLS patch but extends over several hundred nt. In addition, in contrast to replication errors [START_REF] Fujii | DNA replication errors produced by the replicative apparatus of Escherichia coli[END_REF], untargeted mutations assigned to Pol V are refractory to mismatch repair (MMR) correction [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. Indeed, efficient MMR requires association with the replication apparatus [START_REF] Simmons | β Clamp Directs Localization of Mismatch Repair in Bacillus subtilis[END_REF][START_REF] Hasan | Chromosomal directionality of DNA mismatch repair in Escherichia coli[END_REF]. Thus, the observed lack of MMR correction is likely due to the absence of the replication fork context. Such a phenomenon was also observed in yeast where untargeted mutations induced by Pol ζ during gap filling synthesis, are refractory to MMR [START_REF] Kochenova | DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNA[END_REF][START_REF] Lehner | The mismatch repair system promotes DNA polymerase zeta-dependent translesion synthesis in yeast[END_REF]. Taken together, in addition to the intrinsic low fidelity of TLS Pols, ineffectiveness of MMR during post-replicative gap repair exacerbates the genetic instability caused by untargeted mutagenesis.

ALTERNATE ACCESSES OF LOW-AND HIGH-FIDELITY POLS DURING POST-

REPLICATIVE GAP REPAIR

During the overall process of TLS in vivo, Pol V-mediated untargeted mutagenesis was revealed to extend over several hundred nt around a lesion site (Fig. 3C) [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. The intensity of untargeted mutations downstream from the lesion site globally decreases with the distance from the lesion. Surprisingly the decrease is not uniform but exhibits an oscillating (wavy) pattern of valleys and hills suggesting alternate accesses of high-and low-fidelity Pols, respectively [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF].

In principle, any Pol can stochastically access vacant 3'-ends in nascent strands irrespective of whether or not the binding event will be productive, i.e. lead to DNA synthesis. The probability for stochastic access of a given Pol to a vacant T/P will depend upon the relative number of Pol molecules per cell. When Pol V functions, cells are under SOS-induced conditions. Under such conditions, the estimated number of molecules per cell, from Pol I to Pol V, are ≈400, ~350, ~20, ~2,500, and ≈40, respectively [START_REF] Kornberg | DNA Replication[END_REF][START_REF] Kim | Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli[END_REF][START_REF] Sommer | Specific RecA amino acid changes affect RecA-UmuD'C interaction[END_REF][START_REF] Qiu | The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant[END_REF][START_REF] Mchenry | DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits[END_REF]. The total number of high-fidelity Pols (i.e., the classical Pol I to III) is ~770; the relative proportion of "classical Pols", Pol IV and Pol V is thus approximately 23%, 76% and 1.2%, respectively. Despite the high relative abundance of Pol IV (76%), its contribution to untargeted mutagenesis during gap filling synthesis is essentially negligible [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. This observation reflects the above mentioned feature of Pol IV, namely its inefficient functional access to normal T/P. Thereby, it was concluded that Pol V is the main contributor to untargeted mutagenesis [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF] Within the oscillating pattern of untargeted mutations, the apparent length of valleys (i.e., error-free stretches) is relatively short (e.g., a few tens nt) (Fig. 3C) [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF], a size that is compatible with the processivity of classical high-fidelity Pols in the absence of the βclamp [START_REF] Fujii | Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways[END_REF][START_REF] Camps | When pol I goes into high gear: processive DNA synthesis by pol I in the cell[END_REF][START_REF] Fay | Size classes of products synthesized processively by DNA polymerase III and DNA polymerase III holoenzyme of Escherichia coli[END_REF]. This observation suggests that, when classical Pols access 3'-ends of primers, most Pols begin to elongate and dissociate before spontaneously encountering a diffusing sliding β-clamp (Fig. 4A).

The apparent length of the hills (i.e., error-prone stretches) in the oscillating pattern is around 50 nt (Fig. 3C) [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. In contrast to Pol IV on normal T/P (i.e., quick dissociation or one nt elongation) (Fig. 4B), Pol V becomes stabilized by interacting with a 3'-tip of RecA* (i.e., pre-Pol V-fa) thus providing a long enough residency time to interact with a freely sliding β-clamp (i.e., pre-Pol V-fa is converted to Pol V-fa) on normal T/P (Fig. 4C) [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF], as discussed before in the context of TLS (Fig. 2C) [START_REF] Fujii | The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited[END_REF][START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. Pol V-fa begins to elongate on normal T/P with an average processivity of ≈25 nt as determined in vitro (66), a size that is compatible with the apparent length of the observed hills [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. Thereafter, Pol V likely dissociates from the T/P as a sub-complex composed of Pol V and a single RecA monomer (Pol V-dc: Pol V-dissociation complex) [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF][START_REF] Jiang | The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP[END_REF]. Upon dissociation of Pol V and its associated RecA monomer (Pol V-dc), a short ssDNA gap is generated between the vacant 3'-end of the primer and the new 3'-tip of RecA* (Fig. 4C).

What is the Underlying Mechanism of the Alternate Access of Pol V and classical

Pols?

In addition to the untargeted mutation pattern indicating alternate access of lowand high-fidelity Pols described above, intriguingly the overall decline of untargeted mutations with distance from the lesion site is conspicuously moderate [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF] given the numerical superiority of classical Pols over Pol V (the relative ratio, ≈19 : 1). As a minor pathway, a small fraction of the classical Pols encounter a freely sliding β-clamp and synthesize a long stretch in an error-free manner, thus contributing to the overall decline of mutations. (step b in Fig. 5).

What may be the mechanism that allows Pol V to dominate over the classical Pols in spite of its numerical inferiority? If the vacant T/P is formed upon dissociation of Pol V, the short ssDNA gap that is formed may be geometrically unsuitable for functional access of a free Pol V molecule which requires concomitant interaction with T/P and RecA* (step a in Fig. 5). Under such circumstances classical Pols will preferentially access the vacant 3'-end and synthesize, without β-clamp, a relatively short error-free patch (step b in Fig. 5). During synthesis of such short error-free patches by the classical Pols, RecA molecules are dislodged from the 3'-tip of RecA* [START_REF] Fujii | Defining the position of the switches between replicative and bypass DNA polymerases[END_REF][START_REF] Fujii | RecFOR proteins are essential for Pol Vmediated translesion synthesis and mutagenesis[END_REF] forming new 3'-tips of RecA* among which some may exhibit the proper configuration to allow Pol V to simultaneously bind the T/P and the 3'-tip of RecA*. When a classical Pol dissociates, we suggest that its immediate reloading may be sterically hampered by the presence of the RecA filament, which in turn favors loading of Pol V despite its numerical inferiority. Subsequently, Pol V locked onto the T/P (i.e., pre-Pol V-fa) will associate with a freely sliding β-clamp and the resulting functional assembly of Pol V (i.e., Pol V-fa) synthesizes an error-prone patch (step c in Fig. 5), restarting a new cycle (step d in Fig. 5).

The Region Upstream of the Lesion Displays a Dynamic State

Unexpectedly, during the process of Pol V-mediated TLS in vivo, untargeted mutagenesis was also observed in the region upstream from the lesion site albeit with somewhat reduced intensity compared with the downstream region (Fig. 3C) [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. These events likely result from a scenario where exonucleases (with or without helicases) resect the 3'-end of nascent strand in the vicinity of a lesion or of a non-extendable short TLS patch produced by Pol V, thus expanding the single-stranded region upstream from the lesion site, followed by RecA* formation. Subsequently, during gap filling of the region upstream of the lesion, Pol V-fa is formed and induces untargeted mutations. This observation implies that, until late SOS response, the region upstream from the lesion is in a dynamic dsDNA ⇄ ssDNA equilibrium state. During that period, classical Pols are essentially responsible for the fill-in reaction while accuracy of the fill-in reaction will be lower than that of nascent strands at a replication fork due to lack of efficient MMR correction. When Pol V becomes available in late SOS response, the subfraction of these 3'-ends located upstream from the lesion, will be extended in an error-prone way by Pol V. Thus, even before Pol V bypasses a lesion, genome integrity is already compromised.

HOW DOES POL V IMPLEMENT ELONGATION ON RECA-COATED ssDNA?

Pol V Interacts with a Dynamically Formed RecA Filament When Pol V is expressed, all exposed (or SSB-coated) ssDNA regions are converted into RecA-coated ssDNA (RecA*). Globally, the apparent growth of the RecA filament was shown to occur in the 5' → 3' direction on ssDNA [START_REF] Register | The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange[END_REF]. However, RecA filament formation is a highly dynamic process, and individual RecA monomers associate at and dissociate from both ends of the filament at different rates, leading to a net apparent growth in the 5' → 3' direction (Fig. 6) [START_REF] Joo | Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution[END_REF].

Genetic data suggested that a 3'-tip of RecA* is essential for UmuD'2C-induced mutagenesis (i.e., targeted mutagenesis via Pol V), and this RecA function was referred to as third role of RecA [START_REF] Blanco | Influence of RecA protein on induced mutagenesis[END_REF][START_REF] Dutreix | New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis[END_REF][START_REF] Sweasy | RecA protein of Escherichia coli has a third essential role in SOS mutator activity[END_REF]. Thereafter, it was also shown that Pol V requires direct contact at the 3'-tip of RecA* for activation of Pol V in vitro [START_REF] Schlacher | RecA acts in trans to allow replication of damaged DNA by DNA polymerase V[END_REF] and that the ultimate RecA monomer becomes stably associated with Pol V forming a complex composed of Pol V and a RecA monomer [START_REF] Jiang | The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP[END_REF]. It should be stressed that the stability of RecA bound to Pol V is much higher than the stability of the 3'-terminal RecA monomer in RecA*; indeed, 50% of RecA monomer bound to Pol V dissociates in ≈30 min (81), while at the 3'-end of RecA*, RecA monomer dissociates with a half-life (t1/2) of ≈4.3 s (= ln2 / koff (≈0.16 s -1 )).

Biochemical Features of RecA* Filament Formation

The first step in RecA* formation is a nucleation step whereby ≈5 RecA monomers associate on a short stretch of ssDNA (≈20 nt), followed by an extension phase to form RecA* [START_REF] Joo | Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution[END_REF][START_REF] Galletto | Direct observation of individual RecA filaments assembling on single DNA molecules[END_REF][START_REF] Lu | Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity[END_REF]. In other words, the requirement of the nucleation step indicates that individual RecA monomers bound to ssDNA are intrinsically very unstable (e.g., very high koff of RecA) [START_REF] Joo | Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution[END_REF][START_REF] Galletto | Direct observation of individual RecA filaments assembling on single DNA molecules[END_REF][START_REF] Lu | Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity[END_REF][START_REF] Menetski | Interaction of recA protein with singlestranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors[END_REF][START_REF] Arenson | Quantitative analysis of the kinetics of end-dependent disassembly of RecA filaments from ssDNA[END_REF]. In vitro, the efficiency of RecA* formation largely depends upon the nature of the nucleotide analogue that is used as a cofactor [START_REF] Ellouze | Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides[END_REF][START_REF] Wigle | Conformationally selective binding of nucleotide analogues to Escherichia coli RecA: a ligand-based analysis of the RecA ATP binding site[END_REF]. The affinity of RecA for ssDNA decreases in the order, ATP > no cofactor > ADP, and only ATP efficiently supports both co-protease and strand exchange reactions in the context of RecA*. Thus, ATP is seemingly the most physiologically relevant cofactor during RecA* formation in vivo [START_REF] Menetski | Interaction of recA protein with singlestranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors[END_REF][START_REF] Ellouze | Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides[END_REF][START_REF] Roca | Direct Evaluation of a Mechanism for Activation of the RecA Nucleoprotein Filament[END_REF]. During RecA* formation in vitro, the different cofactors induce distinct RecA* filaments. Indeed, RecA-ATP (or RecA-ATP-γS: a poorly hydrolysable ATP analog) and RecA-ADP (or RecA with no cofactor) induce filaments on ssDNA referred to as extended and compressed, respectively [START_REF] Cox | Organized Unidirectional Waves of ATP Hydrolysis within a RecA Filament[END_REF][START_REF] Van Loenhout | Dynamics of RecA filaments on single-stranded DNA[END_REF][START_REF] Boyer | Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination[END_REF]. During de novo filament formation, RecA occupies 3 nt in the extended filament (helical pitch: ≈95 Å) (17) while it occupies 5 nt in the compressed filament (helical pitch: ≈75 Å) [START_REF] Yu | Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible[END_REF].

Conversion from extended to compressed filament was reversibly observed on the same ssDNA molecule in response to changing the nucleotide cofactor, implying that conversion can elastically occur without varying nt occupation of RecA [START_REF] Van Loenhout | Dynamics of RecA filaments on single-stranded DNA[END_REF][START_REF] Nishinaka | Elastic Behavior of RecA-DNA Helical Filaments[END_REF]. We thus suggest that, in the context of physiologically relevant de novo extended RecA-ATP filaments, the event observed in RecA-ADP (or RecA with no cofactor) occupying 5 nt [START_REF] Yu | Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible[END_REF] would be spatially compatible at RecA* extremities (i.e., 3'-and 5'-tips) in a certain situation such as a free RecA-ADP molecule access RecA* extremities.

RecA possesses a DNA-dependent ATPase activity and its turnover number (kcat) for ATP hydrolysis was estimated at ≈0.3 ~ 0.5 s -1 in the presence of ssDNA [START_REF] Brenner | recA protein-promoted ATP hydrolysis occurs throughout recA nucleoprotein filaments[END_REF]. Based on relatively similar range of koff (5'-end, ≈0.12 s -1 ; 3'-end, ≈0.16 s -1 ) (67) and kcat of RecA, dissociation of RecA monomers from ends of RecA* could be linked to ATP hydrolysis, accompanied with a conformational change of RecA (i.e., RecA-ATP → RecA-ADP) [START_REF] Joo | Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution[END_REF][START_REF] Arenson | Quantitative analysis of the kinetics of end-dependent disassembly of RecA filaments from ssDNA[END_REF]. On the other hand, as individual RecA monomers (i.e., not in RecA*) on ssDNA are unstable as described above, dissociation events of such individual RecA monomers on ssDNA are likely not linked to ATP hydrolysis.

The RecA-Fluttering Model

How is Pol V activated on RecA-coated ssDNA? At the outset, Pol V accesses RecAcoated ssDNA leading to form pre-Pol V-fa, a prerequisite for subsequent conversion into Pol V-fa via interaction with the β-clamp (Fig. 7A). The RecA monomer at a 3'-tip of RecA* has greater affinity towards Pol V than towards the next RecA monomer in the RecA* as discussed above. As a consequence, this particular RecA monomer bound to Pol V detaches from RecA* via ATP hydrolysis. The transition of the RecA monomer, from the 3'-tip of RecA* to its stable association with Pol V, is referred to as "RecA-capture". Under single-hit conditions in vitro, Pol V with RecA* (i.e., pre-Pol V-fa) lacks measurable processivity on normal T/P [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. Thus, in the event where RecA-capture occurs in the pre-Pol V-fa state, Pol V with a RecA monomer would immediately dissociate from T/P (i.e., in the form of Pol V-dc), resulting in a non-productive event. On the other hand, in the context of Pol V-fa, owing to the additional stability conferred by the interaction with the β-clamp, the event of RecA-capture leads to a formation referred to as a Pol V functional complex (Pol V-fc: Pol V with a RecA monomer and the β-clamp) (Fig. 7A). The RecA-capture event generates a 3 nt ssDNA gap that cannot support Pol V-mediated DNA synthesis due to steric hinderance; indeed, as mentioned previously, given the change in status of RecA from its ATP to ADP form when it dissociates from ssDNA, re-access of the RecA-ADP onto ssDNA likely requires a 5 nt gap instead of 3 nt. We refer to the sterically blocked form of Pol V as its "immovable state". The transition between Pol V-fa and Pol V-fc would, thus, basically be irreversible (Fig. 7A). Consequently, DNA synthesis by Pol V-fc can only occur upon dissociation of the next 3'-tip of RecA* (koff: ≈0.16 s -1 ), thus generating a 6 nt gap and turning Pol V from an immovable to a movable state (Fig. 7B).

What is RecA-Fluttering? During elongation, the RecA monomer that is part of Pol V-fc likely behaves as either a 5'-tip of RecA* or a free RecA monomer. In either case, given the low binding affinity of a single RecA monomer to ssDNA, we suggest that the RecA monomer in Pol V-fc performs rapid association and dissociation cycles on ssDNA, referred to as "RecA-Fluttering" (Figs. 7C and7D). We suggest that RecA-Fluttering serves to properly position Pol V with the β-clamp on T/P at the nucleotide insertion step during elongation. Indeed, in the absence of RecA under multiple-hit conditions in vitro, Pol V alone has the capacity to produce much longer elongation products than Pol V with the β-clamp at certain natural DNA sequence contexts [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. It implies that the β-clamp impairs proper positioning of Pol V on T/P such as a difficult-to-replicate DNA sequence or a lesion-containing template DNA [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. Immediately after Pol V-fc formation, since most captured RecA monomer would be associated with ADP, this form may be advantageous for Pol V-fc elongation owing to its lower ssDNA binding affinity. Otherwise, due to lack of sliding capacity of RecA on ssDNA [START_REF] Bell | Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA[END_REF][START_REF] Roy | SSB protein diffusion on singlestranded DNA stimulates RecA filament formation[END_REF], a RecA monomer with higher ssDNA binding affinity such as RecA-ATP may modestly behave as an obstacle during Pol V-fc elongation.

While first and second roles of RecA correspond to up-regulation of the mRNA encoding UmuDC and post-translational cleavage of UmuD to UmuD', respectively [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF], we previously suggested that the so-called third role of RecA [START_REF] Blanco | Influence of RecA protein on induced mutagenesis[END_REF][START_REF] Dutreix | New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis[END_REF][START_REF] Sweasy | RecA protein of Escherichia coli has a third essential role in SOS mutator activity[END_REF], corresponds to the stabilization of Pol V on T/P via pre-Pol V-fa (i.e., Pol V with RecA*) formation [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF]. As discussed above, the pre-Pol V-fa complex facilitates the interaction of Pol V with a freely sliding β-clamp, leading to Pol V-fa formation (Fig. 4C). Additionally, another role of RecA allows smooth elongation of Pol V on difficult-to-replicate sequences (e.g., secondary structure) by stretching such sequences that otherwise would lead to strong pause sites (fourth role of RecA) [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF][START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. In contrast to both third and fourth roles that involve RecA in the context of RecA*, its role in Pol V-fc implicates RecA as a monomer. Here, we suggest that in the RecA-Fluttering mode, where the RecA monomer actively functions for proper positioning of Pol V bound to the β-clamp on T/P during the elongation, represents the fifth role of RecA.

Two elongation modes of Pol V-fc. Within the context of a movable Pol V-fc complex, the RecA-Fluttering mode allows to sense whether DNA synthesis is possible when the gap is 6 or more nt long (movable state) or impossible if the gap is only 3 nt (immovable state) (Fig. 7B). Subsequently, elongation on RecA-coated ssDNA is operated via two, non-exclusive elongation modes (discontinuous and continuous modes) depending upon the position of a 3'-tip of RecA* with respect to Pol V-fc. In the discontinuous mode, as a RecA-ADP monomer requires 5 nt for binding to ssDNA, elongation of Pol V-fc on a 6 nt gap would be sterically blocked after only 1 nt incorporation leaving a 5 nt gap (Fig. 7C).

In the subsequent cycle, relying on dissociation of a RecA monomer from the 3'-tip of RecA*, Pol V-fc encounters an 8 nt gap and succeeds to incorporate 3 nt leaving a 5 nt gap in each cycle (Fig. 7C). During elongation, when the RecA monomer in Pol V-fc is converted to RecA-ATP, Pol V-fc could be converted into Pol V-fa via contact of the RecA-ATP with a 3'-tip of RecA*; in such a case, Pol V-fa can be reactivated through the process of RecA-capture as previously mentioned (Fig. 7A). In the continuous mode, when not encountering a blockade (i.e., a 3'-tip of RecA*), Pol V-fc can uninterruptedly elongate (Fig 7D). Accordingly, a DNA patch synthesized by Pol V-fc could be produced by either mode, or a combination of both modes. When the Pol V-fc consumes its residency time on T/P, it dissociates as Pol V-dc (Pol V with a RecA monomer) being a non-productive by-product in the physiological context (Fig. 7E). Based on the average processivity of Pol V-fc (i.e., ≈25 nt), in the case of discontinuous mode, one can estimate that a total of 9 cycles has been performed (steps d to f in Fig. 8).

The apparent koff of Pol V-dc is estimated to be ≈0.012 s -1 as derived from the residency time of Pol V-fc, ≈86 s (= processivity / velocity: ≈25 nt / ≈0.29 nt s -1 ) (66). When Pol V-dc dissociates, it concomitantly creates a 5-8 nt gap depending on whether dissociation occurs at the immovable or the movable state (Fig. 7E). The kinetics of nucleotide cofactor exchange from RecA-ADP to RecA-ATP (e.g., ADP → no cofactor → ATP) as indirectly monitored by ATPase activity seemingly reaches a plateau value at ~10 min [START_REF] Menetski | Interaction of recA protein with singlestranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors[END_REF]. The residency time of Pol V-fc (≈86 s) may thus indicate that during elongation most RecA monomers in Pol V-fc are in the ADP (or no cofactor) state or even after dissociation (Pol V-dc). In addition, a nucleotide cofactor such as ATP-γS was observed to remain in a RecA monomer associated to Pol V over 1 hr [START_REF] Jiang | The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP[END_REF].

The overall view of the RecA-Fluttering model is delineated in Fig. 8. It is noteworthy that, while the average processivity of Pol V-fc is ≈25 nt, the distribution of processive synthesis is relatively broad up to around 100 nt [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. This observation may indicate that processive synthesis entails a combination of discontinuous and continuous elongation modes.

Extremely slow velocity of Pol V-fc. Intriguingly, on normal T/P the velocity of Pol V-fc is over 10-fold slower than that of Pol IV-fc (≈0.29 vs 3-5 nt s -1 ) [START_REF] Wagner | The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity[END_REF][START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. This significant difference likely reflects the unique feature of Pol V during elongation, i.e. its functional association with RecA. Residency times of RecA monomers at the 5'-and 3'-ends of RecA*, deduced from their (koff) -1 values, are estimated to be ≈8.3 s and ≈6.3 s, respectively [START_REF] Joo | Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution[END_REF]. Thus, the residency time of Pol V-fc (≈86 s) is one order of magnitude higher than those of RecA monomers at ends of RecA*. This suggests that, within the residency time of Pol V-fc, multiple RecA monomers (≈14) are committed to dissociate from the 3'-ends of RecA*. Intriguingly, Pol V-fc requires ≈10 s to fill the 3 nt freed upon dissociation of a single RecA monomer, a duration similar to the residency times of a single RecA monomer at the ends of RecA*. Taken together, there appears to be fine tuning between the velocity of Pol V-fc and the dynamic state of RecA at the 3'-end of RecA*.

DISTINCT MODES OF ACTION DURING DNA SYNTHESIS FOR POL IV AND POL V

In contrast to Pol IV-fc (velocity, 3-5 nt s -1 ; processivity, 300-400 nt) [START_REF] Wagner | The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity[END_REF], Pol V-fc exhibits slower velocity (≈0.29 nt s -1 ) and lower processivity (≈25 nt) on normal T/P (66).

Interestingly, the residency times of Pol IV-fc and Pol V-fc on T/P exhibit similar duration, ≈97 s (average from 60-133 s (= 300-400 nt / 3-5 nt s -1 )) and ≈86 s (= ≈25 nt / ≈0.29 nt s - 1 ), respectively. Thus, despite significant differences between Pol IV and Pol V in terms of velocity and processivity on normal T/P, their affinities for T/P are similar, implying that the residency times of both Pol IV and Pol V may largely depend upon their interaction with the β-clamp [START_REF] Wagner | The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity[END_REF][START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF].

Pol IV and Pol V exhibit distinct lesion bypass features, not only in terms of their substrate specificities (minor versus major groove lesions, respectively), but also in the way their processivities are affected by lesions. The processivity of Pol V-fc (≈25 nt on normal T/P) is only moderately reduced by the presence of a lesion (e.g., reduced to ≈18 nt in TLS across an AAF-dG) [START_REF] Fujii | Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation[END_REF]. In contrast, in vitro the processivity of Pol IV-fc during [-ta]-BaP-dG bypass is intriguingly short (around 5 nt) compared to its processivity on normal T/P (300-400 nt) [START_REF] Wagner | The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity[END_REF] suggesting that most of Pol IV's residency time is consumed by the lesion bypass step (S. Fujii, A. Isogawa, R. Fuchs, unpublished data). A similar phenomenon also occurs for Pol II during the bypass of an AAF-dG lesion in the Nar I sequence context, namely its processivity during TLS via a slippage mechanism is only 3 nt, while it is >1,600 nt on normal T/P [START_REF] Fujii | Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways[END_REF][START_REF] Roth | Origin of Mutations Under Selection: The Adaptive Mutation Controversy[END_REF]. It, thus, indicates that Pol IV and Pol V largely differ in their modes of action during lesion bypass. Indeed, the fact that Pol V-fc can deal with a T/P almost as efficiently whether or not a lesion is present can be attributed to its fine positioning by virtue of a RecA monomer in Pol V-fc (i.e., fifth role of RecA). This notion is also supported by the observation that, under multiple-hit conditions in vitro, Pol IV (or Pol II) alone can carry out robust DNA synthesis on both normal and lesioncontaining T/P (N-2-furfuryl-dG for Pol IV; AAF-dG for Pol II) [START_REF] Fujii | Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways[END_REF][START_REF] Jarosz | A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase[END_REF]; in contrast, under multiple-hit conditions in vitro, Pol V alone can perform DNA synthesis on normal T/P but not on a lesion-containing T/P [START_REF] Fujii | The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited[END_REF].

IMPACT OF TLS AND PERSPECTIVES

In E. coli, TLS Pols modestly contribute to survival, but are essential for induced mutagenesis [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF] strongly inferring that, in addition to preventing persistent ssDNA gaps, they represent a major driving force for genetic diversity in both short (e.g., adaptive mutations) or long time range (e.g., evolution) [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF][START_REF] Roth | Origin of Mutations Under Selection: The Adaptive Mutation Controversy[END_REF]100). Biological significance of genetic diversity in short time range is illustrated by, for instance, mutations in the immunoglobulin gene locus (i.e., somatic hypermutation) in higher eukaryote or appearance of antibiotic resistant bacteria in the clinics. The former is driven by TLS Polsmediated targeted and/or untargeted mutagenesis restricted to a localized region in the genome (101). The latter is presumed to result from antibiotic-induced stress that leads to SOS induction causing genome-wide mutagenesis mediated by TLS Pols (102).

Why Many Different Pol V Models have been Proposed over the Years?

Since Pol V was identified, various, sometimes conflicting, models to explain its biochemical features in vitro have been proposed [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF]103,104). We suggest that the subtlety of Pol V's biochemistry stems from two distinct roles that RecA imparts Pol V: i) the ATP-bound form of RecA in RecA* facilitates the interaction of Pol V with a freely sliding β-clamp (third role of RecA) and ii) the ADP-bound form of RecA monomer (fifth role of RecA) allows proper positioning of Pol V with the β-clamp during DNA synthesis.

Complete physiological reconstitution of Pol V's mode of action is thus made difficult by the stepwise involvement of RecA-ATP first followed by RecA-ADP, in addition to the influence of the β-clamp into the Pol V's mode of action. As a consequence, the biochemical properties of Pol V sharply depend upon the experimental conditions such as the nature of the nucleotide cofactor as well as the geometry of the T/P, leading to conflicting results and contributing to the enigmatic feature of Pol V in vitro [START_REF] Fuchs | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF]103,104).

In contrast to previous models, the "RecA-Fluttering" model proposed herein is likely to become a paradigm that reconciles these perplexing longstanding issues (Figs. 7 and8).

Physiological Roles of TLS Pols Beyond the Canonical TLS Pathway

Recent finding that Pol V participates in gap filing synthesis associated with the process of TLS [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF] raises the possibility that TLS Pols such as Pol V participate in any gap filling synthesis irrespective of the origin of the ssDNA gap. For instance, at a late time point during SOS response, a RecA-dependent recombination-mediated repair pathway is activated (step f in Fig. 1) [START_REF] Courcelle | RecA-Dependent Recovery of Arrested DNA Replication Forks[END_REF][START_REF] Courcelle | Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli[END_REF]105). During the process, an ssDNA gap, in all points similar to the gap formed following TLS patch formation, appears (steps c to d vs f to g in Fig. 1). Indeed, it was suggested that TLS Pols potentially participate in homologous recombination in vivo (106). In addition, it was reported that TLS Pols can participate in NER in vivo (107) and confer a competitive fitness advantage during stationary phase (108). Thus, the working place of TLS Pols appears not to be restricted to the sole context of lesion bypass. Consequently, the name of "specialized Pols" instead of TLS Pols may be more appropriate to reflect their diverse functions.

Untargeted Mutagenesis during Post-Replicative Gap Repair as a Beneficial Source of Diversity in Evolution

Classically, genome-wide mutagenesis has been extensively studied through the analysis of mutator mutants (e.g., MMR defective strains) in order to unravel their relative contribution to genome integrity (109). Differently from genome-wide mutators, Pol Vinduced untargeted mutagenesis is restricted to a local area (Fig. 3C) resembling to somatic hypermutation as in immunoglobulin gene loci. Such localized mutagenesis may be beneficial to evolution (110). Moreover, as Pol V-induced untargeted mutations are inefficiently repaired by MMR [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF], one daughter cell will acquire novel genetic information while the another daughter cell retains its original content. It is noteworthy that Pol IV frequently induces -1 frameshift mutations at monotonous base runs [START_REF] Kim | Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA[END_REF] while Pol V essentially induces base substitutions [START_REF] Isogawa | Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Postreplicative Gap Repair[END_REF]. Errors induced by Pol V during gap filling synthesis, in addition to the lack of proofreading function, are not efficiently corrected by MMR, leading to ≥10 4 -fold increase compared to regular DNA replication errors (109, 111). Such features may be physiologically advantageous for generating genetic diversity in short and long time ranges.

SOS-dependent Untargeted Mutagenic Roads

Last but certainly not least, Pol IV is essentially inefficient in gap filling synthesis on normal template DNA as described previously. Therefore, in contrast to post-replicative gap repair associated to Pol V-mediated TLS (Fig. 3C), the mutation spectrum associated to Pol IV-mediated TLS is expected to carry minimum mutagenic load as in Fig. 3B.

Below we describe two situations where, in addition to Pol IV, Pol V becomes involved in completion of the whole process of minor groove lesion bypass. First, some minor groove lesions may require both Pol IV and Pol V for their bypass. Following metabolic activation of BaP in vivo, the ultimate metabolite (diol-epoxide) primarily reacts at the N 2 position of dG (112) forming [+ta]-BaP-dG and its stereoisomer [-ta]-BaP-dG that represent the major and a minor adduct, respectively (113). As mentioned above, in vivo, the [-ta]-BaP-dG adduct is bypassed by Pol IV under normal growth conditions. In contrast, bypass of [+ta]-BaP-dG interestingly requires both Pol IV and Pol V in vivo [START_REF] Seo | Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli[END_REF][START_REF] Napolitano | All three SOSinducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis[END_REF]. Thus, depending upon the precise chemistry and conformation of a minor groove adduct, successful bypass requires collaboration between Pol IV and Pol V. In this situation, when Pol V is required, TLS necessarily occurs in late SOS and post-replicative gap filling becomes error-prone.

Second, when the number of minor groove lesion is high, i.e. exhausting the capacity of rapid Pol IV-mediated bypass, cells enter into late SOS response where Pol V is expressed and can functionally access RecA-coated ssDNA gap. Thus, depending upon the timing of Pol IV-mediated lesion bypass completion, untargeted mutation pattern gradually shifts from largely error-free (Fig. 3B) to error-prone (Fig. 3C) owing to the participation of Pol V.

At the end, considering TLS in vivo from the sole prospect of lesion bypass accompanied by a targeted mutation event, melts down to the famous saying "you cannot see the wood for the trees". As mentioned throughout this review, based upon the overall context of post-replicative gap repair, our vision of TLS pathways should be thoroughly revisited.

Fig. legends FIG 1

 legends1 Fig. legends

FIG 2

 2 FIG 2 Stochastic access of DNA Pols on lesion-containing template DNA. Functional TLS

FIG 3

 3 FIG 3 TLS elicits untargeted mutagenesis during post-replicative gap repair. (A)

FIG 4

 4 FIG 4 Stochastic access of DNA Pols on normal template DNA. (A) Classical Pols can

FIG 5

 5 FIG 5 Pol V-mediated TLS: overall view of the complete post-replicative gap repair

Fig. 1 Fig. 2 51Fig. 3 Fig. 4 53Fig. 5 Fig. 6 55Fig. 7

 1234567 Fig. 1

Fig. 8

 8 Fig. 8

ACKNOWLEDGMENTS

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

The authors declare no competing interests. Conceptualization, S.F.; Writing -original draft, S.F.; Writing -review & editing, S.F. and R.F.

appears. (b) This short gap prevents proper re-access of a Pol V molecule to both the 3'end of the primer and the 3'-tip of RecA*, simultaneously. Thereby, functional access of one of the classical Pols dominates, leading to synthesis of a short error-free patch. Our working model supposes that, when a classical Pol dissociates, it eventually yields a 3'end of primer to which Pol V can bind and simultaneously interact with the 3'-tip of RecA*. monomers in RecA* at 5'-and 3'-ends are essentially the same (5'-end, ≈0.12 s -1 ; 3'-end, ≈0.16 s -1 ), and their association rates (kon) are as follows: 5'-end, ≈0.11 s -1 at 100 nM RecA; 3'-end, ≈0.18 s -1 at 8 nM RecA, leading to the dissociation constants (KD) at 5'-end, ≈100 nM and at 3'-end, ≈8 nM [START_REF] Joo | Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution[END_REF]. Thus, whereas both ends of RecA* are in a highly dynamic state, the significant difference of KD (i.e., the large difference of kon dependent upon concentration of RecA) leads to the apparent directionality of RecA* formation.

FIG 7

The RecA-Fluttering model. RecA molecules are numbered starting at the 3'-end of RecA*. ATP and ADP forms of RecA are shown in light blue and yellow, respectively. If Pol V can carry out elongation (i.e., there is enough free ssDNA space), we will refer to it as an "movable" complex. If not, we will call it "immovable". (A) pre-Pol V-fa (i.e., Pol V with RecA*) is converted into Pol V-fa following interaction with the β-clamp. The 3'-end of dissociates from T/P, ≈9 RecA molecules have been released from the 3'-end of the filament. When Pol V-dc dissociates, Pol V-fc is either in a "immovable" state leaving a 5 nt gap or in a "movable" state leaving a 5-8 nt gap.


Olivier Berné

Alexia Hilaire

We investigate inequalities in access to permanent positions in France, focusing on the hiring stage. We use results from a national survey conducted on behalf of the French society of astronomy and astrophysics (SF2A) aimed at young astronomers holding a PhD obtained in France, and answered by over 300 researchers. We find that women are nearly two times less likely than men to be selected by the (national or local) committees attributing permanent positions (p = 0.06). We also find that applicants who did their undergraduate studies in an elite school ("Grande École"), where women are largely under-represented, rather than in a university, are nearly three times more likely to succeed in obtaining a position (p = 0.0026). Our analysis suggests the existence of two biases in committees attributing permanent positions in astronomy in France: a gender bias, and a form of elitism. These biases against women in their professional life impacts their personal life as our survey shows that a larger fraction of them declare that having children can have a negative effect on their careers. They are half as many as men having children in the sample.

Inequalities in access to permanent positions in astronomy in France

Introduction

Gender inequalities in science have been the focus of numerous studies involving sociology, psychology, economics, etc. Although these inequalities have been well identified for several decades, little progress has been achieved [START_REF] Valian | Why so slow?: The advancement of women[END_REF], in particular in several fields of Science Technology Engineering and Mathematics (STEM) where women remain under-represented [START_REF] Beede | Women in stem: A gender gap to innovation[END_REF], including in physics [START_REF] Glover | Women and scientific employment[END_REF].

Astronomy is also a field where women are under-represented. For instance, there are only 18% of members of the international astronomical union (IAU, the largest organisation of professional astronomers) who are females. This under-representation is often attributed to some forms of discrimination against women astronomers in their careers, one striking example in the discipline being access to observing time on telescopes. Decades ago already it was reported by e.g. Vera Rubin, that access to Mount Wilson observatory for women was a struggle [START_REF] Rubin | margaret burbidge, presidentelect[END_REF]. Recent empirical studies have shown that gender biases in the attribution of telescope time persist at major facilities such as the Atacama Millimeter Array [START_REF] Carpenter | Systematics in the alma proposal review rankings[END_REF], the Hubble Space Telescope [START_REF] Reid | Gender-correlated systematics in HST proposal selection[END_REF], the European Southern Observatory [START_REF] Patat | Gender systematics in telescope time allocation at eso[END_REF], and the NRAO [START_REF] Lonsdale | Gender-related systematics in the nrao and alma proposal review processes[END_REF]. Although women are now better represented in large scientific collaborations in astronomy, only few of them serve as leaders in these teams [START_REF] Lucatello | Diversity and inclusiveness in large scientific collaborations[END_REF], and when it gets to receiving a share of a prize, they can be forgotten [START_REF] Berné | Are women the losers in team cosmology prize?[END_REF]. Regarding the success of women in obtaining permanent/faculty positions, a study by Flaherty [START_REF] Flaherty | The leaky pipeline for postdocs: A study of the time between receiving a phd and securing a faculty job for male and female astronomers[END_REF] has shown for instance that female astronomers leave the academic labour market at a much higher rate then men. This result was however mitigated by another study in which it was found that the rate at which women are hired on long-term positions in astronomy (or closely related fields) in the United States is similar to that of men [START_REF] Perley | Gender and the career outcomes of phd astronomers in the united states[END_REF]. The existence of gender inequalities in the field of astronomy has been covered mostly in the United States, but interest has sparked in other countries (see e.g. [START_REF] Brough | Women in astronomy workshop report[END_REF][START_REF] Matteucci | Women in italian astronomy[END_REF][START_REF] Sedeño | A sociological study of gender and astronomy in spain[END_REF]). Studies for the case of France, which is one important nation in the field, and where only 22% of astronomers holding a permanent position are women lack. In addition, while there are, as we have seen, several studies on gender biases in astronomy, research addressing the articulation between gender and other types of biases at hiring, as well as the impact of such biases on the personal life of female astronomers are scarce. Here we provide an example of such an analysis for the case of France using the results of a survey conducted in 2017 amongst young astronomers in this country.

A national survey on French astronomy

The survey was elaborated by the council of the French Society of Astronomy and Astrophysics (SF2A, www.sf2a.eu), and conducted using an online form. It was aimed at researchers who obtained their PhD in astronomy between 2007 and 2017, in France, but not limited to French nationals. Questions in the survey concerned the profile of the young astronomers, their current status in particular in terms of employment, their applications to permanent positions and the perception of their careers. Profile includes gender, year of PhD, city where PhD was obtained and type of undergraduate studies. Status concerns their current position, either in or outside of research, public or private, permanent or short-term as well as a personal status i.e. whether they have children or not.

A fraction of the survey was dedicated to applications by young astronomers to permanent positions in academia, so as to identify if they had or not applied to permanent positions, of which type (researcher, dep. astronomer, ass. prof), and whether they had succeeded or not. Several questions were devoted to their perception on their careers, in particular if they "[...] believe that having children can be an obstacle in [their] career[s]?". The survey was distributed through national professional astronomy canals, including the newsletter of SF2A.

In total, the survey received 301 answers. This is approximately a third of the total population: about 1000 students obtained their PhD in astronomy Admission rates of PhD holders to permanent positions in academia France between 2007 and 2017 in France according to data collected by the doctoral school of the Paris Observatory. Amongst the 301 answers, 198 self declare as men, 97 as women, and 6 as "do not want to answer". This corresponds to 33% women amongst those who answered, which is very close to the ratio that is observed, independently, of women who defended a PhD in astronomy in France between 2012 and 2018 (32% of PhDs in astronomy in France were defended by women, according to data collected by the doctoral school of the Paris Observatory).

Elitism and gender biases in French astronomy

We studied the admission rates to permanent positions, defined as the ratio between the number people who have applied, to the number of applicants who succeeded in obtaining a position for the three major types of permanent positions available in astronomy in France (see box for details about types of permanent positions in France). It is important to note that hiring at a permanent position in France mostly occurs less then 10 year after PhD. Hence the measured admission rate, which is 16.6%, can be considered as an estimate of the total rate at which the academic system absorbs applicants in astronomy.

We first investigate the admission rates as a function of academic back-ground. In the French (mostly public) education system two systems cohabit in science: universities, which are non-selective and where most students go to, and elite schools i.e. "Grandes Écoles", which are selective and where the "best" high school students go to. The two most prestigious and selective "Grandes Écoles" are the École Normale Suprieure (ENS), and the École Polytechnique, which can be compared in terms of status prestige to Harvard or Cambridge in the "anglo-american education system" [START_REF] Marginson | Global university rankings: where to from here[END_REF], however there are a number of other engineering schools which are also considered as "Grandes Écoles". Based on our data, we find that "Grandes Écoles" graduates have a success rate in their applications to permanent positions of 26.6%, nearly three times that of applicants who did their undergrad studies at the university (10.6%). In order to assess the significance of these results, we perform a χ 2 test on these data. We find an overall probability for the null-hypothesis (fortuitous occurrence of the factor nearly three between university and GE applicants) to be true of p = 0.0026. This result is reminiscent of the type of favouritism linked to the "Grandes Écoles" and part of a general elite reproduction scheme that was identified already decades ago by P. Bourdieu and is described in details in his book "The state nobility" [START_REF] Bourdieu | The state nobility: Elite schools in the field of power[END_REF]. In this work, Bourdieu studies what he calls "School mediated forms of [elite] reproduction" (p 285) to access the "field of power" (p 267) which includes the highest positions in academia (e.g. university professors). He defines this mechanism using a comparison with the forms of cooptation that exist in family-run business : "In the [School mediated mode of reproduction], the academic title becomes a genuine entry pass : the school, in the form of the Grande Ecole -and the corps, a social group that the school produces [...] take the place of the family and family ties, with the cooptation of classmates based on school and corps solidarity, taking over the role played by nepotism and marital ties in business". The results presented here suggest that somehow the mechanisms described by Bourdieu are at play today in the field of French astronomy. In the rest of this paper we will refer to this specific mechanism simply as "elitism". In Fig. 1 we now compare admission rates as a function of gender. It can be seen that, for all types of positions, women have a lower success rate then men. Overall, the success rate for men is twice that of the success rate for women with a significance of p = 0.06. Although this is highly suggestive of a gender bias, it appears to be less important than elitism. A question one can thus ask is whether the observed gender bias is somehow tied to the strong form of elitism we have identified. Indeed, female students are significantly under-represented in "Grandes Écoles" : for instance, there are 16% and 17% women at École Polytechnique and École Normale Superieure, respectively [START_REF] Blanchard | Filles+ sciences= une équation insoluble?. enquête sur les classes préparatoires scientifiques[END_REF], whereas at the university, there are, according to the ministry of higher education, 52% women (more specifically, for the fields of science: 28% in "fundamental science an applications", and 60% in "natural science and biology"). Hence, if recruiting committees in French astronomy tend to favour applicants from "Grandes Écoles", as is the case, this could mechanically reduce the number of women selected at the hiring stage. To untangle the effects of gender and elitism, we define four classes of applicants: women from the university, men from the uni-

Admission of PhD holders to permanent positions in astronomy in France

The role of undergraduate education and gender

Women -university Men -university Women -Grande École Men -Grande École versity, women from "Grandes Écoles", men from "Grandes Écoles", and present their success rates at permanent job applications in Fig. 2. This figure shows that women from "Grandes Écoles" still have two times less chances of success than men from the same schools. Hence, the observed elitism needs to be supplemented by a "genuine" gender bias at hiring to explain the observations in Figs. 1 and2. It is also striking that the difference in success rate between men and women from "Grandes Écoles" is larger than the difference in success rate of male and female university graduates. In a way, it seems that prestige tends to amplify at the same time the absolute success rate and the gender inequality.

Interiorization of gender biases by women

We now investigate how such biases are interiorized (or not) by women in the perception of their careers. In particular, we look at their perception of the compatibility of pursuing a career in academia with maternity. We have asked in the survey if respondents "believe that having children can be an obstacle in their careers?"with 5 possible answers : "yes certainly", "yes most likely", "not really", "not at all", "do not want to answer". In Fig. 3 we present the results to this question as a function of gender. We find that the answer "not at all" is under-represented amongst women while the answer "most likely" is over-represented. The precise opposite trend is observed for men, although with a smaller significance. The overall statistical significance of these trends is however high, with p = 0.001. We can conclude that women fear more the impact of having children on their careers than men. This feeling translates into a real life inequality : in our sample, 27.3% of men have children against [START_REF] Sedeño | A sociological study of gender and astronomy in spain[END_REF] are similar). This shows that gender equity in terms of familial achievements amongst astronomers is elusive, as is observed more generally in academia [START_REF] Mason | Marriage and baby blues: Redefining gender equity in the academy[END_REF]. An additional interesting and perhaps non-intuitive result from our survey is that those who have children fear less the effect of children on their career than those who do not have children.

Conclusion

In conclusion, this study suggests that at least two forms of biases, namely elitism and gender bias exist a the hiring stage in French astronomy. Women declare having more difficulties to conciliate maternity and career. As a consequence, fewer women in astronomy in France have children than men. This could be either because they fear that children have a negative impact on their careers, and/or because those who do have children are somehow "forced" to leave academia.

5 Box: French academic system in the field of astronomy

There are three types of permanent positions in French academia: researcher (chargé' de recherche), assistant professor (maitre de conférence) and deputy astronomer (astronome adjoint). Researchers are hired by the CNRS and do research only. Assistant professors are hired in universities, and teach 196 hours per year in addition to their research. Deputy astronomers are hired in "observatories" by universities and teach 66 hours per year. Senior declinations of these positions are "Director of research", "Astronomer" and "Professor". While it is technically possible to access to these position directly from a non-permanent position, this is in practice very rarely the case, and most often these position are accessed to through career progression (e.g. from CNRS researcher to director of research). Researcher and dep. astronomer positions are attributed on a yearly basis through national competitive examinations by a committee whose members are elected by the community and named by representative of the CNRS and ministry of research and higher education. Ass. prof. positions at universities are attributed locally by a jury composed of internal and external members.

Figure 1 :

 1 Figure 1: Admission rates of applicants (< 10 years after PhD)) to permanent in France.

Figure 2 :

 2 Figure 2: Success rates of four different classes of applicants to permanent positions in astronomy in France.

you believe that having children can be an obstacle in your career ? (Table of standardized residuals of Chi-square test) p-value = 0.001

  Figure3: Results to the question "Do you believe that having children can be an obstacle in your career ?" as a function of gender, shown as a table of standardized residuals. These are the difference between the observed count and expected count in the chi-square hypothesis testing, normalized to the standard deviation σ of the expected count.

	F M	Y e s , c e r t a i n l y M o s t l i k e l y 1 2 Do -2.4 N o t r e a l l y N o t a t a l l N o a n s w e r -1.96 -0.64 -0.2 0.24 0.68 1.12 1.56 2 -1 -2.4 -0.9 -1.52 -1.08 -0.7 -1.4 0.7 1.7 0.6
		.6%
	for women (note that the age distributions in our sample for men and women

Positive (negative) numbers z indicate that an answer is over(under)-represented in a population at z × σ. More precisely, these numbers are Z-scores, with |z| > 1.7 corresponding to a probability p < 0.045 in a one-tailed normal distribution. The indicated p-value below the table concerns the results of the overall χ 2 -test of variables "gender" vs "are children an obstacle".