# Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens

Gregory Hoff, Claire Bertrand, Emilie Piotrowski, Annabelle Thibessard,

Pierre Leblond

► To cite this version:

Gregory Hoff, Claire Bertrand, Emilie Piotrowski, Annabelle Thibessard, Pierre Leblond. Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens. Research in Microbiology, 2017, 168 (1), pp.26-35. 10.1016/j.resmic.2016.07.003. hal-01552147

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# Implication of RuvABC and RecG in Homologous Recombination in *Streptomyces ambofaciens*

Grégory Hoff<sup>a, b</sup>, Claire Bertrand<sup>a, b</sup>, Emilie Piotrowski<sup>a, b</sup>, Annabelle Thibessard<sup>a, b*</sup>, Pierre Leblond<sup>a, b*</sup>

E-mail addresses: gregory.hoff@univ-lorraine.fr (G. Hoff), claire.bertrand@univ-lorraine.fr (C. Bertrand), emilie.piotrowski@univ-lorraine.fr (E. Piotrowski), annabelle.thibessard@univ-lorraine.fr (A. Thibessard)

*Correspondence and reprints, pierre.leblond@univ-lorraine.fr (P. Leblond) *Correspondence and reprints

Corresponding author(*) : Pierre Leblond, pierre.leblond@univ-lorraine.fr, tel +33(0)383684207, Annabelle Thibessard, annabelle.thibessard@univ-lorraine.fr, tel +33(0)383684206

## Abstract

Most bacterial organisms rely on homologous recombination to repair DNA double strand breaks and for the post-replicative repair of DNA single strand gaps. Homologous recombination can be divided in three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a *recA* dependent synaptic step allowing the invasion of an intact copy and the formation of a Holliday junctions and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step. In *Escherichia coli*, branch migration and resolution are performed by the RuvABC complex, but could also rely on the RecG helicase in a redundant manner. In this study, we show that *recG* and *ruvABC* are well-conserved among *Streptomyces*. Δ*ruvABC*, Δ*recG* and Δ*ruvABC ΔrecG* mutant strains were constructed. Δ*ruvABC ΔrecG* is only slightly affected by exposure to DNA damage (UV). We also show that conjugational recombination decreases in absence of RuvABC and RecG but that intra-chromosomal recombination is not affected. These data suggest that RuvABC and RecG are indeed involved in homologous recombination in *S. ambofaciens* and that alternative factors are able to take over Holliday junction in *Streptomyces*.

Key words : *Streptomyces*, homologous recombination, DNA damage, resolvase

<footnote><sup>a</sup>Université de Lorraine, Dynamique des Génomes et Adaptation Microbienne, UMR 1128, Vandœuvre-lès-Nancy, F-54506, France</footnote>

<footnote><sup>b</sup>INRA, Dynamique des Génomes et Adaptation Microbienne, UMR 1128, Vandœuvre-lès-Nancy, F-54506, France</footnote>
**Introduction**

Among all DNA damage that cells have to cope with, double strand breaks (DSB) are the most deleterious ones. They can be induced by various exogenous sources such as chemicals, natural products like mitomycin C or physical forces like desiccation or exposure to ionizing and UV radiations [1-3]. They can also be triggered by collapse of the replication fork after the replication machinery encounters a single-strand nick [4-5] or by the effect of endogenous genotoxic components such as reactive oxygen species (ROS) [6]. In absence of repair, DSBs lead to cell death, but in the course of evolution, two major ways were selected to repair DSBs: Homologous Recombination (HR) and illegitimate recombination including the error-prone Non-Homologous End Joining pathway (NHEJ). In contrast to NHEJ which can directly ligate the free DNA ends after a facultative processing step, HR needs an intact homologous template to repair the break. DNA repair through HR can be described as a sequence of three stages: the pre-synaptic, synaptic and post-synaptic steps. In bacteria, HR was previously well-documented, especially in some model organisms like *Escherichia coli* or *Bacillus subtilis* [7-15]. In the pre-synaptic step, a helicase-nuclease complex binds to the free DNA end and begins to unwind the double strand molecule while extensively degrading both strands until it reaches specific sequences named *chi*. The main factors involved in this step and the nature of the *chi* sequences can vary among organisms [11, 16-17]. The confrontation between the *chi* site and the helicase-nuclease complex causes important changes in the conformation of the latter complex. It leads to the alteration of the 3'-5' exonuclease activity and therefore to the formation of single strand 3' tails on which RecA protein is loaded. The newly formed RecA nucleofilament promotes homologous pairing and strand invasion during the synaptic step [9, 14]. Finally, the post-synaptic step involves the formation, migration and resolution of a four way DNA intermediate named the Holliday junction (HJ), and, at least in *E. coli*, PriA dependent loading of the replicative helicase on the post-synaptic DSB repair intermediate called a D-loop leads to the assembly of a replication fork and couples DSB repair with replication restart [18-20].

A high number of studies concerning the post-synaptic step of HR in bacteria were undertaken in the past decades, but involvement and importance of each factor remain elusive. In *E. coli*, the proteins RuvA, RuvB and RuvC are known to form a tripartite complex able to displace and resolve an HJ [21]. In the presence of ATP, RuvA binds DNA and, together with RuvB, drives the branch migration of the HJ leading to the formation of a DNA heteroduplex [22-23]. In concert with RuvAB, RuvC

resolves the newly formed structure by a dual strand incision [24]. The nicked DNA duplexes can then split up and be restored by DNA ligases [25-26]. HJ migration can also be performed by the RecG helicase [27-29]. Although the role of RuvAB in branch migration and that of RuvABC in resolution of HJs is firmly established, the role of RecG remains more elusive [30]. Recently, RecG was proposed to act on the D-loop formed by the RecA filament invasion rather than on the HJ, and to promote the correct loading of PriA, allowing efficient replication restart and the formation of a viable recombinant molecule [31]. The additive defects conferred by *ruvABC* and *recG* mutations would then result from the fact that HJ branch migration and replication restart from the D-loop are two ways of stabilizing the recombination intermediate. Even though *ruvAB* and *recG* are highly conserved among bacterial genomes [32], the impact of their loss on cell metabolism is variable between organisms. *E. coli* devoid of RuvABC or RecG displays a weak sensitivity to DNA damage and a slight decrease in HR efficiency. In contrast, a double *ΔruvABC ΔrecG* mutant strain shows a marked aggravation of both phenotypes suggesting that the two pathways achieve redundant roles [27]. In *B. subtilis*, the single deletion of either *ruvAB*, or *recG* or *recU* (a functional homologue of *ruvC*) is highly deleterious for cell survival to DNA damage whereas the different combinations (*ΔruvAB ΔrecU*, *ΔruvAB ΔrecG* and *ΔrecG ΔrecU*) of double mutation are lethal [12-33].

*Streptomyces* are Gram-positive soil bacteria belonging to the Actinomycetales order. They are characterised by a complex lifestyle in which a vegetative mycelium gives rise to an aerial mycelium differentiating in spore chains. They are also known to produce a large panel of secondary metabolites. In contrast to most bacterial phyla, they possess a linear chromosomal DNA with a typical replication origin located approximately at the middle of the chromosome [34-35] and terminal proteins attached covalently to the 5' ends of the DNA [36]. The genome of the *Streptomyces* species presents a high plasticity characterized by frequent rearrangements resulting from both homologous recombination triggering chromosomal arm replacement [39-40] and illegitimate recombination leading for instance to chromosomal circularization (for review [39-40]). Extensive DNA amplifications are also frequently encountered on rearranged chromosomes [41] resulting from an unknown mechanism involving homologous recombination [42].

At the moment, little is known about HR factors in *Streptomyces*. While there is no homologue for either *recBCD* of *E. coli* or *addAB* of *B. subtilis* in the *Streptomyces* genomes so far sequenced, these bacteria possess an *adnAB* locus [43] as described in
other actinomycetes [17]. This locus encodes a helicase-nuclease complex meant to play the role of *recBCD* and *addAB* in *E. coli* and *B. subtilis* respectively, which is to process the DNA ends of a DSB and initiate the recombination repair pathway. In contrast to *Mycobacterium tuberculosis*, the deletion of this locus was not possible in *Streptomyces ambofaciens*, strongly suggesting that the encoded function was essential [43]. On the other hand, a mutant deficient for *recA* is viable in *Streptomyces* [44]. Δ*recA* strains are particularly sensitive to UV and mitomycin C exposure and are unable to perform HR in conjugational recombination [44]. Concerning the post-synaptic step of HR, in silico analyses revealed the presence of *ruvABC* and *recG* homologues in *S. coelicolor* and *S. avermitilis* [32], and assessing their role in *S. ambofaciens* HR is the purpose of this study.

## Material and methods

### Bacterial strains and culture conditions.

All strains used in this study are listed in table 1. The DH5α *E.coli* strain is used as conservation host for BACs and plasmids. The ET12567 non methylating strain containing the mobilising pUZ8002 plasmid was used as donor for intergeneric conjugation with *S. ambofaciens*. All *E. coli* strains were grown in Luria-Bertani (LB) medium at 37°C except for the BW25113 pKD20 thermosensitive strain used for PCR targeting, which was grown at 30°C. *S. ambofaciens* ATCC23877 was our reference strain from which the mutant strains derived. All *S. ambofaciens* strains were grown at 30°C on Soya flour mannitol (SFM) plates except for UV assays which were performed on solid Hickey Tresner (HT) medium. Liquid HT medium was used for growth of mycelium before DNA extraction. When necessary, antibiotics were added to the medium at a concentration of 50 µg/µL for apramycin and kanamycin, 25 µg/µL for nalidixic acid and chloramphenicol. For intrachromosomal recombination assays, bacterial lawns were picked using a 2 cm diameter cookie-cutter and spores were harvested after being thoroughly vortexed (3 times 20 s). For conjugational recombination assays, the number of *Streptomyces* spores was calibrated to 10⁸ UFC and added to 2.10⁸ UFC of the *E. coli* donor culture.

### Mutant strain construction.

Mutant strains were constructed by PCR targeting as described by Gust *et al.* 2003 [51]. To summarise, a recombinant BAC containing the locus of interest was transformed in the highly recombinogenic *E. coli* BW25113/pKD20 strain. The target gene was replaced by an apramycin resistance disruption cassette [52] in which an *oriT* sequence as introduced. The modified BAC was then transferred into *S. ambofaciens* ATCC23877 strain using the ET12567/pUZ8002 donor host. Double-crossover (CO) events leading to the replacement of the target gene by the cassette were selected by the sensitivity to kanamycin (loss of the BAC vector part) along with the resistance to apramycin (insertion of the resistance cassette). The disruption cassette flanked by *attL* and *attR* sites was excised in the newly obtained mutant strains after the introduction of pOSV508 a plasmid allowing the expression of *int* and *xis* genes from pSAM2 [52]. Excision was verified by PCR after DNA extraction using a low binding sulfate salt method [53]. All the primers used in this experiment are listed in table 2.

### Ultraviolet (UV) light exposure and viability rates.

Serial dilutions of the spore suspension to be tested were spread on HT plates and exposed to 0, 75 and 150 J.m⁻² UV doses using a 254 nm UV light. Plates were immediately protected from light to avoid photoreactivation and then incubated at 30°C [54]. Surviving colonies were counted after 3 days of growth, and survival rates were calculated in comparison to the same dilutions of unexposed cells of the same culture. For each condition, three biological replicates were carried out.

### Statistical analyses.

For UV light exposure as well as for recombination assays, comparisons were achieved pairwise with a *t* test under R and adjusted with the Bonferroni correction [55].

## Results

### *ruvABC and recG are not essential for Streptomyces development*

In analysis aimed at identification of orthologs of DNA recombination genes, Rocha *et al.* [32] revealed the presence of orthologs of *ruvABC* and *recG* in *S. coelicolor* and *S. avermitilis* genomes. To determine whether *ruvABC* and *recG* loci are well-conserved among *Streptomyces* species, we screened the 36 fully sequenced genomes of *Streptomyces* species (tBLASTN algorithm of the genome database of NCBI). Based on the protein sequence of the genes labelled as *ruvA*, *ruvB*, *ruvC*
and *recG* in *S. coelicolor*, we found an ortholog for each gene in each genome with a minimal identity of 69% for *SCO1519*, 82% for *SCO1518*, 68% for *SCO1520* and 73% for *SCO5556* encoding RuvA, RuvB, RuvC and RecG respectively. The *ruvABC* genes are colocalised and form a potential *ruvCAB* operon. In *S. ambofaciens*, RuvA, RuvB, RuvC and RecG (corresponding to *SAM23877_1570*, *SAM23877_1569*, *SAM23877_1571* and *SAM23877_5320*) have a predicted size of respectively 201, 357, 233 and 745 amino-acids.

We employed the PCR targeting method to construct mutant strains in order to test the involvement of these genes in HR. We generated strains deleted for the *ruvABC* operon or for the *recG* locus as well as double mutant strains. As a HR defective control context, we targeted the *recA* locus. For each genotype, two independent BACs were modified by replacement of the target gene(s) by a resistance cassette and were used to generate two independent mutant strains. All the mutant strains were obtained with classical frequencies showing that none of these genes was essential for growth in laboratory conditions or requested selection for additional suppressor mutations. No specific colony morphology phenotype or sporulation deficiency was observed for the *ΔruvABC* or the *ΔrecG* strains compared to the wild-type (WT) when grown as single colonies along a time course growth of 96 hours, corresponding to the entry in sporulation phase for the WT strain (HT medium, 30°C). In contrast, the double mutant *ΔruvABC ΔrecG* showed a growth phenotype characterised by a slight pigmentation delay (Fig. 1).

### The double Δ*ruvABC* Δ*recG* mutation confers sensitivity to UV light exposure

Spores were exposed to UV at 75 J.m⁻² and 150 J.m⁻² immediately after plating on solid HT medium and survival rate was calculated relatively to unexposed cells. The WT strain showed cell survival rates of 66.45 ± 4.29% and 25.02 ± 2.18% at UV doses of 75 J.m⁻² and 150 J.m⁻² respectively (Fig. 2). The *ΔruvABC* and *ΔrecG* strains showed a sensitivity of 61.93 ± 3.87% and 59.12 ± 4.51% at 75 J.m⁻² and 19.93 ± 1.59% and 17.01 ± 2.12% at 150 J.m⁻² respectively. Even if these values are slightly lower than for the WT, the difference is not statistically significant. In contrast, with sensitivity rates of 43.68 ± 3.52% at 75 J.m⁻² and 11.95 ± 1.66% at 150 J.m⁻², the *ΔruvABC ΔrecG* mutant strains are weakly but significantly more sensitive to UV exposure than the WT (at risk α=0.05). This sensitivity was much less pronounced in comparison to the *ΔrecA* strain which showed a drastic decrease of resistance rates with a value of 0.11 ± 0.08% at the lowest tested dose.

### The double Δ*ruvABC* Δ*recG* mutant is only affected in the formation of conjugational recombinants

To test the ability of the *ΔrecG ΔruvABC* mutant to achieve homologous recombination, we compared the mutant strains and the WT reference for their capacity to form recombinants (Fig. 3 and Fig. 4). For that purpose, we targeted a locus not implicated in DNA recombination processes (namely *SAM23877_1172* encoding the *Streptomyces cellobiose* sensor; [56]). In a first approach (Fig. 3), we introduced by intergeneric conjugation (see material and methods section) the BAC in which *SAM23877_1172* is replaced by an apramycin resistance cassette, then apramycin resistant (Apra<sup>R</sup>) clones were selected for and counted. By this mean, we counted all the recombination events resulting in the integration of the apramycin resistance gene into the chromosome; the most probable being a single cross-over occurring between the insert of the recombinant vector and its homologous counterpart on the chromosome (recombination within the H1 or H2 region; the event within H1 is represented on Fig. 3A). By doing so, we also accounted the minor proportion of recombinants that directly underwent a double cross-over leading to chromosome allele replacement (recombination within H1 and H2, Fig. 3A).

Apra<sup>R</sup> clones resulting from single or double cross-over events were counted in each genetic context and compared to the WT (Fig. 3B). The ratio of Apra<sup>R</sup> clones obtained in *ΔruvABC* and *ΔrecG* contexts was 28.71 ± 5.16% and 19.82 ± 3.64% respectively relative to the WT showing that conjugational recombination efficiency was reduced on average 3.5-fold and 5-fold in *ruvABC* and in *recG* defective mutants respectively. In the homologous recombination deficient *recA* mutant, no Apra<sup>R</sup> clone could be observed in the same experimental conditions. When both loci are deleted, the recombination efficiency was further reduced to 7.4 ± 2.38% of the WT reference. These results demonstrated that *ruvABC* and *recG* are involved in conjugational recombination. Since the recombination deficient phenotype is aggravated in the double *ΔruvABC ΔrecG* mutant strains compared to single mutants, we concluded that RuvABC and RecG may be redundant for the formation of recombinants. However, it should be noticed that in the absence of both RuvABC and RecG, HR is still effective suggesting that alternative proteins can catalyse migration and resolution of Holliday junctions in this double mutant.

Since our observations followed an intergeneric *E. coli-Streptomyces* conjugation, differences in the conjugational capacities of the WT and the *ΔruvABC ΔrecG* genetic backgrounds may be
responsible for the decrease in recombinants. To
rule out this possibility, we set up conjugation
experiments using the conjugative and integrative
plasmid pDYN6902 (derived from pIJ6902); its
chromosomal integration occurs by site-specific
recombination and is therefore independent from
HR [49]. This vector shares the same replication
origin than pBeloBAC11 used to construct the BAC
library [50] and is thus expected to have an
equivalent copy number in the donor E. coli strain.
Since its integration is very efficient, we monitored
the conjugation frequency of the conjugative and
integrative pDYN6902 plasmid in the mutant strain
and compared it to the WT. There was no
significant difference between both strains with
conjugational rates of 1.2·10⁻⁴ and 9.7·10⁻⁵ for the
WT and Δ*ruvABC ΔrecG* strains respectively.

We further set up a second experiment to compare
the frequency of homologous recombination in the
different mutants (Fig. 4). In this approach, we
assayed the frequency of gene replacement
(SAM23877_1172 by the apramycin resistance
cassette). This step corresponds to the formation of
a crossing-over occurring between the long tandem
repeats resulting from the integration of the
recombinant BAC into the genome. The second
cross-over event removes the vector DNA including
the kanamycin resistance gene leading to a Apra<sup>R</sup>
Kan<sup>S</sup> phenotype from a Apra<sup>R</sup> Kan<sup>R</sup> background.
We used an experimental approach inspired from
the classical Luria and Delbrück fluctuation test
[57]. The idea was to test the occurrence of the
second cross-over in independent samples of WT
and Δ*ruvABC ΔrecG* bacterial populations from
clones that have integrated the recombinant vector
by a single cross-over. Twenty samples of bacterial
lawns (picked using a cookie-cutter, see material
and methods section) and thus corresponding to
equal subpopulations were harvested for WT and
Δ*ruvABC ΔrecG* strains in which the first cross-
over happened (Apra<sup>R</sup> Kan<sup>R</sup> transconjugants).
Spore suspensions from these lawn samples were
titrated and grown on HT medium supplemented
with only apramycin to retain the presence of the
resistance cassette but without counter-selecting the
formation of the second crossing-over. Plates were
further replica-plated on HT medium supplemented
with both apramycin and kanamycin to determine
the frequency of Apra<sup>R</sup> Kan<sup>S</sup> clones among the
Apra<sup>R</sup>.

For both genetic contexts, at least one Apra<sup>R</sup> Kan<sup>S</sup>
clone was counted in each of the 20 samples of
populations revealing that the second cross-over
could occur at an equivalent frequency (Fig. 4B).
Furthermore, Apra<sup>R</sup> Kan<sup>S</sup> clones were observed at
frequencies which were not significantly different
(risk α = 0.05): 13.38 ± 4.95% and 11.67 ± 4.98%
for the WT and the ΔruvABC ΔrecG respectively.
According to Luria and Delbrück, this reveals that

intra-chromosomal homologous recombination is
not significantly affected in the Δ*ruvABC ΔrecG*
double mutant strain compared to WT.

These data show that the deficiency in RuvABC
and RecG does not significantly affect
intrachromosomal homologous recombination in S.
ambofaciens while it decreases the capacity to form
recombinants after a conjugational transfer.

Discussion

Alternative homologous recombination post-
synaptic proteins functioning in Streptomyces

RuvABC resolvasome and RecG branch migration
enzyme are assumed to act as alternative proteins
for the post-synaptic step of homologous
recombination in E. coli [8, 58]. While RuvAB and
RecG are able to perform branch migration after HJ
formation, the endonuclease RuvC can resolve the
four way structure and restore the two double strand
homologs [59]. In addition to branch migration of
HJs, in vitro RecG can act on a variety of
structures, and promote a variety of reactions, so
that to date its role in vivo remains undetermined
[30]. In this study, we found a high level of
conservation of ruvABC and recG in all
Streptomyces genomes sequenced so far. We
studied the involvement of both in HR in our
bacterial model S. ambofaciens. For this purpose,
we measured the sensitivity to UV irradiation (a
DNA damaging agent), we quantified homologous
recombination frequency after conjugational
transfer and homologous recombination between
tandemly repeated sequence on the chromosome.
The ΔruvABC ΔrecG is more sensitive to UV
irradiation than wild-type cells and in a ΔruvABC
ΔrecG mutant strain we detected a 7.4 fold decrease
of HR efficiency in conjugational recombination. In
contrast, no difference in intra-chromosomal
recombination was observed between the mutant
and the WT strain. The latter result shows that
proteins may compensate for the deficiencies of
RuvABC resolvasome and RecG enzyme. An open
question is how to explain the significant difference
in HR measured by conjugation and by
intramolecular recombination assay. In
conjugational HR, the substrate DNA (exogenote)
is transferred from E. coli to Streptomyces as a
single strand DNA. In E. coli, it is well-known that
persistent single strand DNA leads to the induction
of the SOS system [60]. This favours interspecies
recombination through the increasing expression of
recA and ruvAB [60]. Hence, we can extrapolate
and hypothesise that the single-stranded transfer of
the BAC DNA from E. coli to Streptomyces also
stimulates the expression of these two
recombination enzymes. Similarly, UV irradiation
is known to induce the SOS response. Increased expression of RecA may stimulate the first steps of HR, increasing the need for the enzymes that act at later steps. In contrast, intrachromosomal recombination would not be promoted by the SOS-induction of RecA and the RuvABC/RecG defect could be compensated by the basic expression of alternative resolvases.

*Migration and resolution of HJ, a high redundant function?*

In the bacterial models *E. coli* and *Bacillus subtilis* RuvABC/RecU and RecG are known to be the main factors of the HR post-synaptic steps [7, 12, 33, 58]. Unexpectedly, the simultaneous inactivation of *ruvABC* and *recG* confers a mild phenotype in response to DNA damage and in HR efficiency on *S. ambofaciens*, suggesting the existence of alternative ways of processing post-synaptic molecules. In *E. coli*, when the late stages of HR are blocked (e.g. through *ruvABC* mutations), the loss of *uvrD*, known to be involved in mismatch and nucleotide excision repair, is lethal [61]. In addition, *E. coli* *UvrD* was recently shown to be able to unwind HJ *in vitro* and it could therefore have a redundant role with RuvAB and RecG [62]. In *S. ambofaciens* genome, two *uvrD* homologues can be detected. Another candidate for branch migration could be a *recA* paralog known as *sms* or *radA* that is well conserved among all bacterial genomes sequenced thus far [63]. In *E. coli*, measurements of conjugational recombination suggest a redundant function for *radA/sms* with the other branch migration factors [64]. Recently, a study showed that RadA/Sms is able to perform branch migration but it is suspected to act differently than RuvAB and RecG since RadA/Sms displays no helicase activity [65].

For the HJ resolution activity, an alternative candidate can also be suggested. Aravind *et al.* [66] identified the YqgF family as sharing homologous and structural relationships with the RuvC family, suggesting that its members, predicted resolvases, could function as an alternative to RuvC. While preparing this manuscript, Nautiyal *et al.* [67] reported *in vitro* analyses carried out on RuvX (the mycobacterial member of the YqgF family). They revealed that this protein has affinity for HJs and displays HJ cleavage activity. *Mycobacteria* and *Streptomyces* both belong to the actinobacteria and an ortholog to RuvX can be identified in the *Streptomyces* genomes. Altogether, these data support the idea that RuvX is a serious candidate for resolving HJs as an alternative to RuvC in *S. ambofaciens*. It would be interesting to investigate the involvement of the aforementioned putative alternative factors in the recombination process in *Streptomyces*.

**Conflict of interest**

The authors declare no conflict of interest.

**Acknowledgements**

This work was supported by the French National Research Agency program Streptoflux (ANR Blanc 0096_01), by ANR through the Laboratory of Excellence ARBRE (ANR-12- LABXARBRE-01) and by the Région Lorraine. We are grateful to Dr. Bénédicte Michel (CNRS, Gif-sur-Yvette, France) for helpful discussions and critical reading of the manuscript.

**References**

[1] Bonura T, Smith KC. Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of *Escherichia coli* K-12. J Bacteriol 1975;121:511-517.

[2] Ward JF. Nature of lesions formed by ionizing radiation. DNA damage and repair, Springer; 1998; p. 65-84.

[3] Vignard J, Mirey G, Salles B. Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up. Radiother Oncol 2013;108:362-369.

[4] Michel B, Ehrlich SD, Uzest M. DNA double-strand breaks caused by replication arrest. EMBO J 1997;16:430-438.

[5] Mehta A, Haber JE. Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb Perspect Biol 2014;6:a016428-a016428.

[6] Li Y, Trush MA, Yager JD. DNA damage caused by reactive oxygen species originating from a copper-dependent oxidation of the 2-hydroxy catechol of estradiol. Carcinogenesis 1994;15:1421-1427.

[7] Taylor AF. Movement and resolution of Holliday junctions by enzymes from *E. coli*. Cell 1992;69:1063-1065

[8] Lloyd RG, Sharples GJ. Processing of recombination intermediates by the RecG and RuvAB proteins of *Escherichia coli*. Nucleic Acids Res 1993;21:1719-1725.

[9] Kowalczykowski SC, Eggleston AK. Homologous pairing and DNA strand exchange proteins. Annu Rev Biochem 1994;63:991-1043

[10] Myers RS, Stahl FW. Chi and the RecBCD enzyme of *Escherichia coli*. Annu Rev Genet 1994;28:49-70.

[11] Chedin F, Ehrlich SD, Kowalczykowski SC. The *Bacillus subtilis* AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro. J Mol Biol 2004;298:7-20.

[12] Sanchez H, Kidane D, Reed P, Curtis FA, Castillo Cozar M, Graumann PL, et al. The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in *Bacillus subtilis*. Genetics 2005;171:873-883.

[13] Chedin F, Handa N, Dillingham MS, Kowalczykowski SC. The AddAB helicase/nuclease forms a stable
complex with Its cognate sequence during translocation.
J Biol Chem 2006;281:18610-18617.

[14] Cox MM. Motoring along with the bacterial RecA protein. Nat Rev Mol Cell Biol 2007;8:127-138.

[15] Dillingham MS, Kowalczykowski SC. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol Mol Biol Rev 2008;72:642-671.

[16] Bianco PR, Kowalczykowski SC. The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'. Proc Natl Acad Sci U S A 1997;94:6706-6711.

[17] Sinha KM, Unciuleac M-C, Glickman MS, Shuman S. AdnAB: a new DSB-resecting motor-nuclease from mycobacteria. Genes Dev 2009;23:1423-1437.

[18] Kogoma T, Cadwell GW, Barnard KG, Asai T. The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair. J Bacteriol 1996;178:1258-1264.

[19] Sandler SJ, Samra HS, Clark AJ. Differential suppression of priA2: Kan phenotypes in *Escherichia coli* K-12 by mutations in *priA*, *lexA*, and *dnaC*. Genetics 1996;143:5-13.

[20] Sandler SJ, Marians KJ. Role of PriA in replication fork reactivation in *Escherichia coli*. J Bacteriol 2000;182:9-13.

[21] Grove JI, Harris L, Buckman C, Lloyd RG. DNA double strand break repair and crossing over mediated by RuvABC resolvase and RecG translocase. DNA Repair 2008;7:1517-1530.

[22] Tsaneva IR, Müller B, West SC. ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of *E. coli*. Cell 1992 69:1171-1180.

[23] Parsons CA, Tsaneva I, Lloyd RG, West SC. Interaction of *Escherichia coli* RuvA and RuvB proteins with synthetic Holliday junctions. Proc Natl Acad Sci U S A 1992;89:5452-5456.

[24] Connolly B, Parsons CA, Benson FE, Dunderdale HJ, Sharples GJ, Lloyd RG, et al. Resolution of Holliday junctions *in vitro* requires the *Escherichia coli* *ruvC* gene product. Proc Natl Acad Sci U S A 1991;88:6063-6067.

[25] Dunderdale HJ, Benson FE, Parsons CA, Sharples GJ, Lloyd RG, West SC. Formation and resolution of recombination intermediates by *E. coli* RecA and RuvC proteins. Nature 1991;354:506-510.

[26] Iwasaki H, Takahagi M, Shiba T, Nakata A, Shinagawa H. *Escherichia coli* RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J 1991;10:4381.

[27] Lloyd RG, Buckman C. Genetic analysis of the *recG* locus of *Escherichia coli* K-12 and of its role in recombination and DNA repair. J Bacteriol 1991;173:1004-1011.

[28] Lloyd RG, Sharples GJ. Dissociation of synthetic Holliday junctions by *E. coli* RecG protein. EMBO J 1993;12:17.

[29] Meddows TR, Savory AP, Lloyd RG. RecG helicase promotes DNA double-strand break repair. Mol Microbiol 2004;52:119-132.

[30] Lloyd RG, Rudolph CJ. 25 years on and no end in sight: a perspective on the role of RecG protein. Curr Genet. 2016;doi:10.1007/s00294-016-0589-z.

[31] Azeroglu B, Mawer JSP, Cockram CA, White MA, Hasan AMM, Filatenkova M, et al. RecG directs DNA synthesis during double-strand break repair. PLoS Genet

2016;12:e1005799.

[32] Rocha EPC, Cornet E, Michel B. Comparative and evolutionary analysis of the bacterial homologous recombination systems. PLoS Genet 2005;1:e15.

[33] Sanchez H, Carrasco B, Cozar MC, Alonso JC. *Bacillus subtilis* RecG branch migration translocase is required for DNA repair and chromosomal segregation. Mol Microbiol 2007;65:920-935.

[34] Bentley SD, Chater KF, Cerdeno-Tarraga A-M, Challis GL, Thomson NR, James KD, et al. Complete genome sequence of the model actinomycete *Streptomyces coelicolor* A3 (2). Nature 2002;417:141-147.

[35] Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, et al. Complete genome sequence and comparative analysis of the industrial microorganism *Streptomyces avermitilis*. Nat Biotechnol 2003;21:526-531.

[36] Huang C-H, Tsai H-H, Tsay Y-G, Chien Y-N, Wang S-L, Cheng M-Y, et al. The telomere system of the *Streptomyces* linear plasmid SCP1 represents a novel class. Mol Microbiol 2007;63:1710-1718.

[37] Fischer G, Wenner T, Decaris B, Leblond P. Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of *Streptomyces ambofaciens*. Proc Natl Acad Sci U S A 1998;95:14296-14301.

[38] Uchida T, Miyawaki M, Kinashi H. Chromosomal arm replacement in *Streptomyces griseus*. J Bacteriol 2003;185:1120-1124.

[39] Volff J-N, Altenbuchner J. Genetic instability of the *Streptomyces* chromosome. Mol Microbiol 1998;27:239-246.

[40] Thibessard A, Leblond P. Subtelomere plasticity in the bacterium *Streptomyces*. In Subtelomeres, Springer, 2014, p. 243-258.

[41] Leblond P, Demuyter P, Moutier L, Laakel M, Decaris B, Simonet J-M. Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in *Streptomyces ambofaciens*. J Bacteriol 1989;171:419-423.

[42] Volff J-N, Viell P, Altenbuchner J. Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in *Streptomyces lividans*. Mol Gen Genet 1997;253:753-760.

[43] Zhang L, Nguyen HC, Chipot L, Piotrowski E, Bertrand C, Thibessard A, Leblond P. The *adnAB* locus, encoding a putative helicase-nuclease activity, is essential in *Streptomyces*. J Bacteriol 2014;196:2701-2708.

[44] Huang T-W, Chen CW. A *recA* null mutation may be generated in *Streptomyces coelicolor*. J Bacteriol 2006;188:6771-6779.

[45] Pinnert-Sindico, S. Une nouvelle espèce de *Streptomyces* productrice d'antibiotiques: *Streptomyces ambofaciens* n. sp. caractères culturaux. Ann Inst Pasteur 1954;87:702-707.

[46] Hanahan D. Studies on transformation of *Escherichia coli* with plasmids. J Mol Biol 1983;166:557-580.

[47] MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil T. Analysis of *Streptomyces avermitilis* genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 1992;111:61-68.

[48] Datsenko KA, Wanner BL. One-step inactivation of
chromosomal genes in *Escherichia coli* K-12 using PCR products. Proc Natl Acad Sci U S A 2000;97:6640–6645.

[49] Thibessard A, Bertrand C, Hiblot J, Piotrowski E, Leblond P. Construction of pDYN6902, a new *Streptomyces* integrative expression vector designed for cloning sequences interfering with *Escherichia coli* viability. Plasmid 2015;82:43–49.

[50] Choulet F, Aigle B, Gallois A, Mangenot S, Gerbaud C, Truong C, et al. Evolution of the terminal regions of the *Streptomyces* linear chromosome. Mol Biol Evol 2006;23:2361–2369.

[51] Gust B, Challis GL, Fowler K, Kieser T, Chater KF. PCR-targeted *Streptomyces* gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 2003;100:1541–1546.

[52] Raynal A, Karray F, Tuphile K, Darbon-Rongere E, Pernodet J-L. Excisable cassettes: new tools for functional analysis of *Streptomyces* genomes. Appl Environ Microbiol 2006;72:4839–4844.

[53] Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical *Streptomyces* genetics. John Innes Foundation, Norwich, United Kingdom, 2000.

[54] Volff JN, Vandewiele D, Simonet JM, Decaris B. Ultraviolet light, mitomycin C and nitrous acid induce genetic instability in *Streptomyces ambofaciens* ATCC23877. Mutat Res 1993;287:141-156

[55] Bonferroni CE. Il calcolo delle assicurazioni su gruppi di teste, In Studii in Onore del Profesor S. O. Carboni, 1935, p. 13-60.

[56] Schlösser A, Jantos J, Hackmann K, Schrempf H. Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader *Streptomyces reticuli*. Appl Environ Microbiol 1999;65:2636–2643.

[57] Luria SE, Delbrück M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 1943;28:491-511.

[58] Whitby MC, Ryder L, Lloyd RG. Reverse branch migration of Holliday junctions by RecG Protein: a new mechanism for resolution of intermediates in recombination and DNA Repair. Cell 1993;75:341-350.

[59] Müller B, West SC. Processing of Holliday junctions by the *Escherichia coli* RuvA, RuvB, RuvC and RecG proteins. Experientia 1994;50:216–222.

[60] Matic I, Rayssiguier C, Radman M. Interspecies gene exchange in bacteria: the role of SOS and mismatch repair systems in evolution of species. Cell 1995;80:507-515.

[61] Magner DB, Blankschien MD, Lee JA, Pennington JM, Lupski JR, Rosenberg SM. RecQ promotes toxic recombination in cells lacking recombination intermediate-removal proteins. Mol Cell 2007;26:273–286.

[62] Carter AS, Tahmaseb K, Compton SA, Matson SW. Resolving Holliday junctions with *Escherichia coli* UvrD helicase. J Biol Chem 2012;287:8126–8134.

[63] Lin Z, Kong H, Nei M, Ma H. Origins and evolution of the *recA/RAD51* gene family: evidence for ancient gene duplication and endosymbiotic gene transfer. Proc Natl Acad Sci U S A 2006;103:10328–10333.

[64] Beam CE, Saveson CJ, Lovett ST. Role for radA/sms in recombination intermediate processing in *Escherichia coli*. J Bacteriol 2002;184:6836–6844.

[65] Cooper DL, Boyle DC, Lovett ST. Genetic analysis of

*Escherichia coli* RadA: functional motifs and genetic interactions. Mol Microbiol 2015;95:769–779.

[66] Aravind L, Makarova KS, Koonin EV. Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. Nucleic Acids Res 2000;28:3417–3432.

[67] Nautiyal A, Rani PS, Sharples GJ, Muniyappa K. *Mycobacterium tuberculosis* RuvX is a Holliday junction resolvase formed by dimerisation of the monomeric YqgF nuclease domain. Mol Microbiol 2016;00:00-00.
<table><caption>**Table 1:** Strains, plasmids and BACs used in this study</caption><thead><tr><th>Strains, plasmids and BACs</th><th>Characteristics</th><th>Sources</th></tr></thead><tbody><tr><td><strong>Streptomyces</strong></td><td></td><td></td></tr><tr><td><em>S. ambofaciens</em> ATCC 23877</td><td>Used as a wild-type strain</td><td>[45]</td></tr><tr><td><em>S. ambofaciens</em> ATCC 23877 Δ<em>ruvABC</em></td><td>Deletion of the <em>ruvABC</em> locus</td><td>This study</td></tr><tr><td><em>S. ambofaciens</em> ATCC 23877 Δ<em>recG</em></td><td>Deletion of the <em>recG</em> locus</td><td>This study</td></tr><tr><td><em>S. ambofaciens</em> ATCC 23877 Δ<em>recG</em>Δ<em>ruvABC</em></td><td>Deletion of the <em>recG</em> and the <em>ruvABC</em> loci</td><td>This study</td></tr><tr><td><em>S. ambofaciens</em> ATCC 23877 Δ<em>recA</em></td><td>Deletion of the <em>recA</em> locus</td><td>This study</td></tr><tr><td><strong>E. coli</strong></td><td></td><td></td></tr><tr><td>DH5α</td><td>F<sup>-</sup> φ<em>dlacZ</em> Δ<em>M15</em> endA1 supE44 thi-1 <em>relA1</em> gyrA96 deoR nupG Δ(<em>lacZYA-argF</em>)U169 λ-<em>hsdR17(rκm<sub>k</sub><sup>+</sup>)phoA</em> <em>relA1</em> <em>dam-13::Tn9</em> dcm <em>cat</em> <em>tet</em> <em>hsdM</em> <em>hsdR</em> zjj-201::<em>Tn10</em> tra neo RP4</td><td>[46]</td></tr><tr><td>ET12567/pUZ8002</td><td>K12 derivative Δ<em>araBAD</em>, Δ<em>rhaBAD</em></td><td>[47]</td></tr><tr><td>BW25113/pKD20</td><td>λ-RED (<em>gam</em>, <em>bet</em>, <em>exo</em>), <em>bla</em>, <em>araC</em>, <em>rep101<sup>ts</sup></em></td><td>[48]</td></tr><tr><td><strong>Plasmids/BACs<sup>a</sup></strong></td><td></td><td></td></tr><tr><td>BAA8H12Δ<em>SAM1569-SAM1571</em></td><td>Δ<em>chl::kan</em> Δ<em>ruvABC::aac3(IV)-oriT</em></td><td>This study</td></tr><tr><td>BAB10F9Δ<em>SAM5320</em></td><td>Δ<em>chl::kan</em> Δ<em>recG::aac3(IV)-oriT</em></td><td>This study</td></tr><tr><td>BAB24H12Δ<em>SAM5320</em></td><td>Δ<em>chl::kan</em> Δ<em>recG::aac3(IV)-oriT</em></td><td>This study</td></tr><tr><td>BAB20B5Δ<em>SAM5473</em></td><td>Δ<em>chl::kan</em> Δ<em>recA::aac3(IV)-oriT</em></td><td>This study</td></tr><tr><td>BAB13C7Δ<em>SAM1172</em></td><td>Δ<em>chl::kan</em> Δ<em>cebR::aac3(IV)-oriT</em></td><td>This study</td></tr><tr><td>pDYN6902</td><td><em>E. coli</em> to <em>Streptomyces</em> conjugative and integrative shuttle vector</td><td>[49]</td></tr></tbody></table>

<footnote><sup>a</sup> all original BACs arise from [50]</footnote>
<table><caption>**Table 2:** Primers used in this study</caption><thead><tr><td>Primer name</td><td>Sequence (5'-3')</td><td>Characteristics</td></tr></thead><tbody><tr><td>kanDchl1</td><td>AGGTTCCAACTTTCACCATAATGAAATAAGATCA CTACCGCCCAAGTCCGCCCATCTC</td><td>Replacement of chl by neo</td></tr><tr><td>kanDchl2</td><td>TGTCGTGCCAGCTGCATTAATGAATCGGCCAACG CGAACCATGAGATCCCCGCGCTGGA</td><td>Replacement of chl by neo</td></tr><tr><td>ruv_L3</td><td>GTGCGCGTACTGGGGGTGGACCCGGGACTGACCC GTCGCGGTGCCTCTTCGTCCCGAAGCAACTTG</td><td>ruvABC deletion</td></tr><tr><td>ruv_R3</td><td>GGACGCGGCGGGGTGAGGCCGAGGTGCGCCCAC GCGGCCGGGCGCGCTTCGTTCGGGACGAAGAGG</td><td>ruvABC deletion</td></tr><tr><td>ruv11</td><td>CGAGGAATTCGGGTCGGTACTTCCTTCACA</td><td>Verification of ruvABC deletion</td></tr><tr><td>ruv12</td><td>GCGAGAATTCCATGGAGAAGGTCGACGCGT</td><td>Verification of ruvABC deletion</td></tr><tr><td>D_recG_F</td><td>TGGCGTGGTGTGCAATGGATCTCGTGCCCGCACT GCGAGTGCCTCTTCGTCCCGAAGCAACTTG</td><td>recG deletion</td></tr><tr><td>D_recG_R</td><td>TCAGCCCTTCTCCAGGTACTGCTCCCTCTCCTCGT CCAGGCGCGCTTCGTTCGGGACGAAGAGG</td><td>recG deletion</td></tr><tr><td>V_recG_F</td><td>CGCTGCTCATCGGCGTG</td><td>Verification of recG deletion</td></tr><tr><td>V_recG_R</td><td>TGTGTTCCGTTCAGTCTGGC</td><td>Verification of recG deletion</td></tr><tr><td>DrecA_F</td><td>GTGAAGCGATCGAATCAAGCAAACCGGGTGGAAC CCATGGTGCCTCTTCGTCCCGAAGCAACTTG</td><td>recA deletion</td></tr><tr><td>DrecA_R</td><td>CCCAGTCGGTTCGTCGTGTCACGGGTCAGCTCTTG GCTGCGCGCGCTTCGTTCGGGACGAAGAGG</td><td>recA deletion</td></tr><tr><td>recA11</td><td>CCGGGAATTCGGACGTACGCTCGGAGGTGC</td><td>Verification of recA deletion</td></tr><tr><td>recA12</td><td>TGGCGAATTCGTCTCCTCCGCTCACGCTCG</td><td>Verification of recA deletion</td></tr><tr><td>SaCebRmut_F</td><td>CCGCAGAGTGCGGGGTCTCGACGGGAGGCGGAGC CGATGATCGCGCGCGCTTCGTTCGGGACGAA</td><td>Deletion of SAM23877_1172</td></tr><tr><td>SaCebRmut_R</td><td>CCGCACGGCACCCCGTGCCGCCCGCTCCCCCAC CCTCAATCTGCCTCTTCGTCCCGAAGCAACT</td><td>Deletion of SAM23877_1172</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>**Fig.1:** Phenotype of *S. ambofaciens* ATCC 23877 WT and *ΔruvABC ΔrecG* strains after 96 hour growth on HT medium at 30°C. In comparison to WT, a *ΔrecG ΔruvABC* strain shows a slight pigmentation delay.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig.2:** Survival rates of WT, *ΔruvABC*, *ΔrecG*, *ΔruvABC ΔrecG* and *ΔrecA* strains after UV exposure at 75 J.m⁻² (dark grey) and 150 J.m⁻² (light grey). This experiment was performed in triplicate. The asterisk (*) indicates a significant difference with WT (t-test) at risk α=0.05.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig.3:** Conjugational recombination efficiency in *S. ambofaciens*. A) Representation of single or double cross-over events occurring between *Streptomyces* chromosome and a homologous DNA transferred by intergeneric conjugation. The *neo* gene confers resistance to kanamycin and *aac* stands for *aac(3)IV* which encodes the aminoglycoside 3-N-acetyltransferase and confers resistance to apramycin. B) Proportion of single and double cross-over events represented by the proportion of Apra<sup>R</sup> clones obtained after conjugation in Δ*ruvABC*, Δ*recG*, Δ*ruvABC ΔrecG*, Δ*recA* and WT strains. Values of the mutant strains were normalised to that of WT. The letters a, b, c and d indicate the different statistical groups (t-test) at risk α=0.05.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<table><caption>**Fig.4:** Intrachromosomal recombination efficiency in *S. ambofaciens*. A) Representation of a second cross-over event occurring within the chromosome after a single cross-over event. B) Proportion of second cross-over events represented by the proportion of Apra<sup>R</sup> Kan<sup>S</sup> clones in the 20 populations obtained after a single cross-over event in WT and ΔruvABC ΔrecG backgrounds.</caption><thead><tr><th colspan="4">WT</th><th colspan="4">ΔruvABC ΔrecG</th></tr><tr><th>Population</th><th>Total Apra<sup>R</sup></th><th>Apra<sup>R</sup> Kan<sup>S</sup></th><th>Apra<sup>R</sup> Kan<sup>S</sup> %</th><th>Population</th><th>Total Apra<sup>R</sup></th><th>Apra<sup>R</sup> Kan<sup>S</sup></th><th>Apra<sup>R</sup> Kan<sup>S</sup> %</th></tr></thead><tbody><tr><td>1</td><td>151</td><td>18</td><td>11.92%</td><td>1</td><td>87</td><td>21</td><td>24.14%</td></tr><tr><td>2</td><td>80</td><td>17</td><td>21.25%</td><td>2</td><td>76</td><td>9</td><td>11.84%</td></tr><tr><td>3</td><td>50</td><td>5</td><td>10.00%</td><td>3</td><td>91</td><td>6</td><td>6.59%</td></tr><tr><td>4</td><td>92</td><td>7</td><td>7.61%</td><td>4</td><td>131</td><td>10</td><td>7.63%</td></tr><tr><td>5</td><td>49</td><td>5</td><td>10.20%</td><td>5</td><td>135</td><td>11</td><td>8.15%</td></tr><tr><td>6</td><td>43</td><td>6</td><td>13.95%</td><td>6</td><td>103</td><td>11</td><td>10.68%</td></tr><tr><td>7</td><td>39</td><td>3</td><td>7.69%</td><td>7</td><td>151</td><td>13</td><td>8.61%</td></tr><tr><td>8</td><td>134</td><td>9</td><td>6.72%</td><td>8</td><td>143</td><td>20</td><td>13.99%</td></tr><tr><td>9</td><td>91</td><td>13</td><td>14.29%</td><td>9</td><td>130</td><td>19</td><td>14.62%</td></tr><tr><td>10</td><td>34</td><td>6</td><td>17.65%</td><td>10</td><td>86</td><td>10</td><td>11.63%</td></tr><tr><td>11</td><td>49</td><td>7</td><td>14.29%</td><td>11</td><td>136</td><td>18</td><td>13.24%</td></tr><tr><td>12</td><td>78</td><td>6</td><td>7.69%</td><td>12</td><td>107</td><td>19</td><td>17.76%</td></tr><tr><td>13</td><td>27</td><td>6</td><td>22.22%</td><td>13</td><td>143</td><td>18</td><td>12.59%</td></tr><tr><td>14</td><td>110</td><td>17</td><td>15.45%</td><td>14</td><td>33</td><td>2</td><td>6.06%</td></tr><tr><td>15</td><td>87</td><td>12</td><td>13.79%</td><td>15</td><td>118</td><td>8</td><td>6.78%</td></tr><tr><td>16</td><td>58</td><td>8</td><td>13.79%</td><td>16</td><td>160</td><td>7</td><td>4.38%</td></tr><tr><td>17</td><td>29</td><td>7</td><td>24.14%</td><td>17</td><td>51</td><td>4</td><td>7.84%</td></tr><tr><td>18</td><td>32</td><td>3</td><td>9.38%</td><td>18</td><td>169</td><td>32</td><td>18.93%</td></tr><tr><td>19</td><td>43</td><td>5</td><td>11.63%</td><td>19</td><td>34</td><td>4</td><td>11.76%</td></tr><tr><td>20</td><td>36</td><td>5</td><td>13.89%</td><td>20</td><td>185</td><td>30</td><td>16.22%</td></tr></tbody><tfoot><tr><td colspan="4">Mean</td><td colspan="4">Mean</td></tr><tr><td colspan="4">Variance</td><td colspan="4">Variance</td></tr><tr><td colspan="4">Standard deviation</td><td colspan="4">Standard deviation</td></tr><tr><td colspan="4">σ=4.95%</td><td colspan="4">σ=4.98%</td></tr></tfoot></table>