# Copy-choice illegitimate DNA recombination revisited.

E. d'Alençon, M. Petranovic, B. Michel, P. Noirot, Anne Aucouturier, M.

Uzest, S.D. Ehrlich

► To cite this version:

E. d'Alençon, M. Petranovic, B. Michel, P. Noirot, Anne Aucouturier, et al. Copy-choice illegitimate DNA recombination revisited. The EMBO Journal, 1994, 13 (11), pp.2725 - 2734. 10.1002/j.1460-2075.1994.tb06563.x. hal-05070603

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<figure><img src="image_1.png" /></figure>
# Copy-choice illegitimate DNA recombination revisited

E.d'Alençon, M.Petranovic¹, B.Michel, P.Noirot, A.Aucouturier, M.Uzest and S.D.Ehrlich

Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas cedex, France and ¹Institut Ruder Boskovic, Bijenicka 54, 41000 Zagreb, Republic of Croatia

Communicated by S.D.Ehrlich

Nearly precise excision of a transposon related to Tn10 from an *Escherichia coli* plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.

**Key words:** copy-choice DNA recombination/*Escherichia coli*

## Introduction

Genome rearrangements are important in evolution (Ohno, 1970; Anderson and Roth, 1977; Campbell, 1979), medicine (Yunis, 1983; Yen et al., 1987; Cooper and Schmidtke, 1989) and biotechnology (Ehrlich et al., 1986). They often result from recombination between sequences of little or no homology, termed 'illegitimate recombination' [Franklin, 1971; for a review see Anderson (1987), Ehrlich (1989) and Meuth (1989)]. This process is not well understood, despite its importance, possibly because it can be carried out in a number of ways. Two broad classes of illegitimate recombination events can nevertheless be distinguished. One is mediated by enzymes which cut and join DNA, such as topoisomerases, site-specific nucleases and proteins which initiate rolling circle replication. Errors of these enzymes can lead to deletions, inversions and formation of cointegrates. Many factors might affect the error rate, as shown for deletions which result from the activity of the phage M13 replication protein where pausing of the replication forks and intense transcription play an important role (Michel et al., 1989; Bierne et al., 1991; Vilette et al., 1992).

Another class of illegitimate recombination events involves short directly repeated sequences that are not known to be recognized by enzymes which cut and join DNA. These events can be frequent and have been reported in prokaryotes (Farabaugh et al., 1978; Albertini et al., 1982; Lopez et al., 1984; Conrad et al., 1992), as well as lower and higher eukaryotes, including man (Efstratiadis et al., 1980; de Zamaroczy et al., 1983; Nalbantoglu et al., 1986; Wallace,

1992; Morris and Thacker, 1993). Recombination between short repeats often generates deletions, but it can also lead to gene duplications (Edlund and Normark, 1981; Whoriskey et al., 1987; Shyamala et al., 1990) and the formation of cointegrates (Bashkirov et al., 1988; Dempsey and Dubnau, 1989; Schiestl et al., 1993). Deletion formation has been studied more thoroughly than other rearrangements and it has been shown that features such as repeat length and GC content, as well as the proximity of palindromic sequences, affect its frequency (DasGupta et al., 1987; Williams and Müller, 1987; Peeters et al., 1988; Singer and Westlye, 1988; Weston-Hafer and Berg, 1989, 1991; Pierce et al., 1991; Sinden et al., 1991). However, in contrast to the first class of events, it is still not known whether recombination between short repeats involves breakage and joining of DNA or whether it is a copy-choice process.

There is ample evidence from transformation experiments in prokaryote and eukaryote systems that DNA linearized *in vitro* can be recircularized *in vivo*, and that the process frequently involves short homologous sequences (Conley et al., 1986; Grzesiuk and Carroll, 1987; Roth and Wilson, 1988). This indicates that recombination between short direct repeats can take place by a break/join process, a model for which comprises the following steps: (i) a double-stranded break occurs between the repeats; (ii) an exonuclease resects the resulting ends and exposes short complementary sequences; and (iii) these sequences anneal and the ends are repaired by polymerase and ligase activities. However, several lines of genetic evidence suggest that short direct repeats might recombine by errors of DNA replication; a tip of a growing DNA chain could slip from one repeat to the other and be used as a primer for further DNA synthesis (Egner and Berg, 1981; Albertini et al., 1982). Such a model was first suggested to explain frame-shift mutations (Streisinger et al., 1966) and is conceptually related to copy-choice recombination (Lederberg, 1955). It can explain the unexpectedly high frequency of recombination between 4 bp repeats relative to 9 bp repeats in an *Escherichia coli* plasmid (Weston-Hafer and Berg, 1989, 1991), the orientation-dependent difference in deletion frequency of a palindrome from an *E.coli* plasmid (Trinh and Sinden, 1991) and the stimulation of excision of Tn5 in DNA polymerase mutants of *Saccharomyces cerevisiae* (Gordenin et al., 1992). To find out whether break/join or copy-choice recombination takes place *in vivo* we decided to examine the feature which distinguishes most clearly the two: namely whether the recombinant molecules contain DNA from the parental molecules, which is expected for the former but not for the latter. Such an approach was followed in a previous study (Brunier et al., 1988), but the report was retracted when it was established that the results of the experiments conducted by the senior author were fabricated (Ehrlich and Michel, 1990).

In the present work transposon excision was used as a
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Structure of the parental and recombinant plasmids. (A) Schematic representation of pHV857. pBR322 and pC194 sequences, a thin line and an empty box, respectively; Ap<sup>R</sup>, Tc<sup>R</sup> and Cm<sup>R</sup> genes, heavy arrows; M13 replication origin, a hatched box. The direction of replication from the pBR322 and M13 replication origins is indicated by arrows outside the circle. The site of insertion of miniTn10-Km is indicated by Tn. (B) Schematic representation of miniTn10-Km. The Km<sup>R</sup> gene of Tn903 is represented by a line and inverted repeats of Tn903 by empty arrows and of IS10R by filled arrows. Sequence of the IS10R ends is shown. The imperfect direct repeats of 24 bp are underlined by thin arrows. (C) Top: sequence of the region upstream of the Ap<sup>R</sup> gene and the beginning of the gene prior to miniTn10 insertion. The ribosome binding site (RBS) and the start codon (START) are boxed. The vertical arrow indicates the site of insertion of miniTn10. Bottom: sequence of the same region after transposon insertion. The transposon is represented by a box, the duplicated 9 bp sequence is underlined. (D) Sequence of the same region in recombinant plasmids, resulting from nearly precise excision involving the first (top) or the last (bottom) 10 bp of the 24 bp direct repeats. Only one DNA strand is represented in all cases.</figcaption></figure>

model. Insertion of transposons such as Tn5 and Tn10 duplicates 9 bp of the target site and their excision deletes the transposon and one of the duplications (Egner and Berg, 1981; Foster et al., 1981). A related process, called nearly precise excision, occurs by recombination between imperfect repeats of 24 bp present close to the ends of Tn10 (Ross et al., 1979). Neither type of excision requires the transposase and both are stimulated by long inverted repeats (Egner and Berg, 1981; Foster et al., 1981). The transposon was carried on an *E.coli* plasmid in which rolling circle replication could be induced. We show that nearly precise excision was stimulated greatly by rolling circle replication and we present evidence that it did not entail the transfer of DNA from the parental to the recombinant molecule. This result supports a copy-choice model of recombination between short direct repeats.

## Results

### Experimental system

To study transposon excision we used an *E.coli* plasmid, designated pHV857 (Figure 1A), which carries a composite transposon, miniTn10-Km (Way et al., 1984). The plasmid contains two replication origins, that of plasmid ColE1 and that of bacteriophage M13. The former is active constitutively and maintains pHV857 in the host cell, while the latter functions only in the presence of the phage replication proteins and leads to production of single-stranded plasmid DNA by rolling circle replication.

The miniTn10-Km transposon is composed of the 1 kb kanamycin resistance (Km<sup>R</sup>) gene of Tn903 and inverted repeats of 411 bp, part of which (363 bp) derives from Tn903 and part (78 bp) from IS10R (Figure 1B). Its insertion between A and T of the start codon of the ampicillin resistance (Ap<sup>R</sup>) gene of pHV857 duplicates 9 bp upstream of the T (Figure 1C), which inactivates the gene, presumably

by blocking its transcription because the gene is still preceded by a functional ribosome binding site and has a start codon (Figure 1C). The gene can be reactivated by nearly precise excision of the transposon, defined as recombination between imperfect direct repeats of 24 bp (the central 4 bp are not repeated), present within the IS10-derived inverted repeats (Figure 1D; Ross et al., 1979). The precise excision, by recombination between the 9 bp duplications generated by transposon insertion, occurs at much lower frequency than the nearly precise excision (Ross et al., 1979) and has not been observed in this study. Plasmids resulting from excision are referred to as recombinant plasmids and cells carrying these plasmids as recombinant cells.

### Infection by f1 stimulates transposon excision

To test whether activation of the M13 origin affects the rate of transposon excision, Hfr3.OSOΔ64 cells carrying pHV857 were infected with phage f1 and allowed to grow for 3 h at 37°C. Samples were withdrawn at 30 min intervals and spread on plates containing Ap or Tc. A typical experiment is shown in Figure 2A. The number of total (Tc<sup>R</sup>) and recombinant (Ap<sup>R</sup>) cells increased exponentially during most of the experiment, but that of recombinant cells increased approximately three times faster. The proportion of recombinant cells increased therefore 1000-fold within five generations of infection (Figure 2B) and reached ~70% overnight (not shown). Similar results were obtained with another strain, JJC41, which is an AB1157 F' derivative, although the increase was generally somewhat lower (~100-fold in five generations). As expected, the proportion of recombinant cells did not increase if the culture was not infected with f1 (Figure 2B).

The proportion of recombinant cells could increase for several reasons, such as: (i) their faster growth compared with parental cells; (ii) enrichment of pre-existing recombinant plasmids; or (iii) generation of novel
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** The proportion of recombinant cells increases upon f1 infection. (A) Exponentially growing Hfr3.OSOΔ64 cells carrying plasmid pHV857 were infected with f1 and the number of total and recombinant cells was determined by plating appropriate dilutions of the culture on media supplemented with tetracycline (●) and ampicillin (■), respectively. The indicated doubling times were calculated from the linear regression curves, excluding the last value (the cell's growth was slowing down at that time). (B) The proportion of recombinants. Stars refer to uninfected cultures (–f1), other symbols to f1-infected cultures (+f1; five experiments with independent clones are shown, different symbols refer to different clones). The proportion of recombinants increased from 1.8 ± 1.2×10⁻⁴ to 1.6 ± 0.2×10⁻¹ in five generations in infected culture.</figcaption></figure>

recombinant plasmids. To test the first possibility we used an isogenic couple of strains: a leucine auxotroph JJC41 and an isogenic leucine prototroph. The parental plasmid was introduced into the two strains and a recombinant clone was isolated from each by selection for ampicillin resistance. Two exponentially growing cultures were prepared, containing either a mixture of 90% parental *leu*⁻ cells and 10% recombinant *leu*⁺ cells or a mixture of 90% parental *leu*⁺ cells and 10% recombinant *leu*⁻ cells. The cultures were infected with phage f1 and the proportion of the two cell types was determined immediately and after 3 h of growth by plating cells on medium containing leucine and replicating the colonies on medium lacking leucine. This allowed us to calculate the doubling time of the parental and recombinant cells. The parental *leu*⁺ and *leu*⁻ cells doubled every 35.9 and 36.7 min, respectively, whereas the recombinant *leu*⁺ and *leu*⁻ cells doubled every 39 and 39.1 min, respectively. The values for the parental cells might be slightly underestimated because recombinants were being formed

during the experiment (Figure 2). However, the values observed with parental and recombinant cells are not significantly different, which shows that the latter have no growth advantage over the former.

The proportion of ApR cells could also increase by enrichment of pre-existing recombinant plasmids. For instance, recombinant plasmids could co-exist with parental plasmid in many uninfected cells but at a copy number too low to confer the resistance, and the phage infection could lead to a progressive increase of their copy number. To test this possibility, a single copy of the recombinant plasmid was introduced by f1-mediated transduction into exponentially growing Hfr3.OSOΔ64 cells, which either harboured pHV857 or were plasmid-free (a phage stock prepared on recombinant cells, containing 4×10¹¹ phage/ml and 2×10⁹ transducing particles/ml, was used). In both cases ~1% of cells were ApR immediately after infection, as determined by plating on Ap or Tc supplemented media (this proportion was high enough to mask the ApR cells present in the plasmid-carrying culture before infection). Doubling time of ApR cells was the same in both cultures for a further 120 min (not shown), suggesting that even a single copy of recombinant plasmid can be detected very rapidly in the presence of the parental plasmid. Taken together, the above experiments indicate that phage infection does not lead to enrichment of pre-existing recombinant cells or plasmids, but to the formation of novel recombinant plasmids.

### Plasmid DNA contained in recombinant cells

Double-stranded plasmid DNA was prepared from uninfected and infected recombinant Hfr3.OSOΔ64 cells and analysed by electrophoresis, intact and cut by several restriction enzymes (not shown). Uninfected cells contained mainly heterodimeric plasmids composed of one parental and one recombinant molecule, while the infected cells contained mostly recombinant monomers. Transformation experiments confirmed these observations, because 75–95% of ApR transformants obtained with DNA from the former cells were resistant to kanamycin, while all ApR transformants obtained with DNA from the latter cells were sensitive to kanamycin. The formation of heterodimeric plasmids during recombination has been reported previously (Mazin *et al.*, 1991), and could be a consequence of incompatibility between the parental and recombinant replicons. Rolling circle replication, induced by phage infection, should resolve dimeric plasmids into monomers (Dotto and Horiuchi, 1981) which, being incompatible, then segregate.

Recombinant plasmids were characterized further by fine restriction mapping (not shown). All plasmids analysed (50) resulted from nearly precise excision. Two isolates from uninfected cells and four from infected cells were analysed by sequencing of the region within which the transposon was inserted. Nearly precise excision involved in three cases the first and in three cases the last 10 bp of the 24 bp imperfect repeat (Figure 1D).

### The proportion of recombinant molecules increases during infection

To verify that the increase of the proportion of recombinant cells is due to the appearance of recombinant plasmids, we extracted total DNA from Hfr3.OSOΔ64 cells and used it to transform *E.coli*-competent cells selecting for ApR or
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Proportion of recombinant plasmids increases upon f1 infection. Total DNA was prepared from Hfr3.OSOΔ64 cells carrying plasmid pHV857 at different times after infection and used to transform MC1061-competent cells to Ap<sup>R</sup> and Tc<sup>R</sup>.</figcaption></figure>

Tc<sup>R</sup>. The proportion of Ap<sup>R</sup> transformants increased exponentially with time (from 5×10<sup>-4</sup> at 30 min to 5.5×10<sup>-2</sup> at 180 min; Figure 3), in parallel with the increase in the proportion of recombinant cells in the same experiment (from 1×10<sup>-3</sup> to 1×10<sup>-1</sup>). This indicates that recombinant molecules are generated constantly upon infection.

**Phage replication proteins stimulate transposon excision**

Three proteins are required for f1 replication. The gene II protein initiates rolling circle replication by introducing a nick into the phage (+) replication origin and terminates replication by joining the ends of the displaced DNA strand. The gene X protein, encoded by the 3' end of gene II, is required for accumulation of single-stranded DNA. Finally, the gene V protein binds to single-stranded DNA and prevents its conversion to a double-stranded form (Model and Russel, 1988). If transposon excision was stimulated by pHV857 rolling circle replication, the three phage proteins should be sufficient for stimulation. To test this prediction we used plasmids which carried genes II and V under the control of an IPTG-inducible promoter. Proteins II and X were provided in trans (from a pSC101 derivative carrying a *lacUV5*–gene II fusion, designated pSCgpII), whereas protein V was provided in cis (a *lacUV5*–gene V fusion was cloned into the *PvuII* site of pHV857, yielding a plasmid designated pHV857gpV). To control the expression of the phage genes the *lacI*<sup>q</sup> repressor gene was cloned on pACYC184 (yielding plasmid pAClacI<sup>q</sup>). The three plasmids (pSCgpII, pHV857gpV and pAClacI<sup>q</sup>) are compatible and can be maintained in the same cell.

To measure the rate of transposon excision JJC40 cells harbouring pHV857gpV, pSCgpII and pAClacI<sup>q</sup> were grown in liquid medium to exponential phase and the ratio

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Single-stranded plasmid DNA is synthesized upon f1 infection. Total DNA was extracted from a constant amount of Hfr3.OSOΔ64 cells containing pHV857 at different times after infection and analysed by Southern hybridization using pBR322 as a probe. (A) DNA was denatured before transfer to nitrocellulose membrane and both double- and single-stranded DNA are visible. (B) DNA was not denatured before transfer and only single-stranded DNA is visible. Positions of the supercoiled parental plasmid double- (Pd) and single-stranded (Ps) monomer and recombinant double- (Rd) and single-stranded (Rs) monomer are indicated. Lane 1, uninfected parental cells. Parental cells at different times after infection: 0 min, lane 2; 30 min, lane 3; 60 min, lane 4; 90 min, lane 5; 120 min, lane 6; 150 min, lane 7; and 180 min, lane 8. Lane 9, uninfected recombinant cells. Lane 10, recombinant cells 60 min after infection.</figcaption></figure>

of Ap<sup>R</sup>:Tc<sup>R</sup> cells was determined by plating. The ratio was equal to 2.5×10<sup>-4</sup> and was constant for at least 6 h in the absence of IPTG, but increased exponentially to 7×10<sup>-1</sup> during that time if IPTG was added. Therefore, transposon excision can be stimulated by the phage replication proteins in the absence of other phage products. Both gene II and gene V are required for efficient stimulation of transposon excision, because in the absence of gene II (cells containing pHV857gpV and pAClacI<sup>q</sup>) or gene V (cells containing pHV857, pSCgpII and pAClacI<sup>q</sup>) the ratio of Ap<sup>R</sup>:Tc<sup>R</sup> cells remained constant or increased only slightly (from 10<sup>-4</sup> to 10<sup>-3</sup>, respectively) during 10–15 generations of cell growth in the presence of IPTG. The *E. coli* single-stranded DNA binding protein, overproduced from a pACYC-derived plasmid, could not replace the gene V product (not shown).

To verify that the same excision event occurs upon induction of the phage replication genes and phage infection, the DNA content of 12 Ap<sup>R</sup> clones was analysed by electrophoresis (not shown). In all cases four different plasmids were found, corresponding to the three starting molecules (pHV857gpV, pSCgpII and pAClacI<sup>q</sup>) and to a plasmid 1.8 kb smaller than pHV857gpV. The last plasmid was purified from seven clones by transformation and selecting for Ap<sup>R</sup> and was analysed by several restriction enzymes. The results indicated that it was formed by nearly precise excision of the transposon (not shown). This conclusion was confirmed by sequencing the relevant region of two plasmids.

**Single-stranded DNA synthesis is required for stimulation of transposon excision**

To test whether single-stranded DNA synthesis is required for stimulation of transposon excision several experiments were carried out.

First, it was established that phage infection induces
synthesis of pHV857 single-stranded DNA. For this purpose total DNA was prepared from a constant number of Hfr3.OSOΔ64 cells at different times after phage infection and analysed by Southern hybridization using pBR322 as a probe (Figure 4). Only the double-stranded parental plasmid was present in uninfected cells but the single-stranded form appeared 30 min after infection (migrating at two positions, possibly because hairpin structures formed on a fraction of molecules by annealing of the transposon inverted repeats) and its amount increased throughout the experiment. The amount of double-stranded DNA increased ~2-fold, probably due to activity of the plasmid-carried M13 replication origin. Recombinant DNA was visible in a double-stranded form after 90 min of infection, and in a single-stranded form after 150 min.

Second, a 695 bp segment carrying the M13 replication origin was deleted from pHV857. The ratio of recombinant and parental cells in an uninfected culture of Hfr3.OSOΔ64 carrying this plasmid was 6×10⁻⁵, close to that observed for pHV857, indicating that the absence of this segment does not affect transposon excision. However, this ratio did not change significantly (to 4×10⁻⁵) upon overnight infection with f1, whereas it reached ~70% when the cells harboured pHV857, which shows that stimulation of excision requires the presence of the M13 replication origin.

Third, to show that stimulation of excision is due to synthesis of plasmid single-stranded DNA we made use of the fact that f1-dependent DNA synthesis requires a specific *E.coli* helicase, Rep. Plasmids pHV857gpV, pSCgpII and pAClacIq were introduced into isogenic JJC41 *rep*⁺ and JJC42 *rep*⁻ cells. Overnight cultures were grown without or with IPTG, which induces synthesis of the phage replication proteins (see above), and the ratio of ApR and TcR cells was determined. As expected, the ratio was low in all cultures grown without IPTG (between 1.1×10⁻⁴ and 6.6×10⁻⁴). It was not significantly higher in *rep*⁻ cells grown with IPTG (between 5.5×10⁻⁴ and 11×10⁻⁴), but was much higher (6.3×10⁻¹) in *rep*⁺ cells grown with IPTG.

**Transposon excision does not entail transfer of DNA from parental to recombinant plasmid**

To examine whether parental DNA becomes a part of recombinant plasmid molecules upon transposon excision, it was density labelled by growing cells in 'heavy' medium, the cells were transferred to 'light' medium and single-stranded DNA synthesis was induced by phage infection. After several generations, required to accumulate a high proportion of recombinants, most of the original DNA strands should be present in molecules composed of one heavy and one light strand. These molecules should contain a high proportion of recombinants if the DNA is transferred from parental to recombinant plasmids upon transposon excision, or a low proportion of recombinants if it does not.

Exponentially growing Hfr3.OSOΔ64 cells carrying pHV857 were first cultured in the presence of [³H]thymidine to radioactively label their DNA and thus facilitate detection of plasmids in the subsequent analysis. They were then transferred to a heavy medium, containing bromodeoxyuridine (BUdR) and [³H]thymidine, and grown for 1 h (~1.5 generations) to density label their DNA. (Longer growth in the presence of BUdR decreased cell viability.) Plasmid DNA was prepared from part of the

culture and analysed by centrifugation in CsCl gradients (the remainder of the culture was processed as described in the following paragraph). Three types of molecule were observed having two (HH), one (HL) or no (LL) density labelled strands, as judged from their buoyant density, and carrying ~25, ~55 and ~20% of total radioactivity, respectively (Figure 5A). This indicates that, during density labelling, some plasmids replicated twice (HH), most replicated once (HL) and some did not replicate at all (LL), as expected for a labelling period of ~1.5 generations and random replication of plasmid molecules (Bazaral and Helinski, 1970). It is noticeable that the molecules containing one or two heavy strands give broader peaks than those containing no heavy strands, which is probably due to a heterogeneous distribution of BUdR in different strands (Meselson *et al.*, 1957). Southern analysis indicated that the three types of molecules contain similar proportions of different plasmid forms (monomers, dimers and higher oligomers) and confirmed that most of the molecules are of the HL type. The three types of molecule were purified by another cycle of CsCl gradient centrifugation (Figure 5A1-3). Southern analysis revealed (i) accumulation of slowly migrating forms of HH molecules (Figure 5A1), probably due to nicking (this can be seen occasionally in other experiments, see below) and (ii) some cross-contamination in side fractions, the peak fractions being pure. Specific activity of H and L strands was determined using the purified HH and LL peak fractions, and found to be ~2-fold higher for the H strand. This must be due to a higher proportion of [³H]thymidine incorporated during cell growth in heavy than in light medium. As a consequence, H and L strands contained 62 and 38% of total radioactivity, respectively.

The remaining cells, grown in heavy medium (see above) and washed to eliminate BUdR, were infected or not by phage f1 and grown for 3 h in a light medium, devoid of BUdR. Their DNA was prepared and analysed by centrifugation, as above. Two peaks were observed with DNA from the uninfected cells (Figure 5B), containing HL and LL molecules and carrying ~45 and ~40% of radioactivity, respectively. They were separated by a region of intermediate density (I) which carried ~15% of radioactivity. HH molecules were no longer present, indicating that all plasmids had replicated at least once. Southern analysis indicated that most of the molecules were of the LL type, as expected from prolonged growth in light medium. Parental monomers, dimers and some higher oligomers were detected. The two peaks and the intermediary region were subjected to another cycle of centrifugation to isolate pure molecules of the different types (HL, I and LL; Figure 5B1-3). Southern analysis indicated that some nicking took place during purification of the HL molecules (Figure 5B1). The two peaks (HL and LL; Figure 5B1 and B3) were not highly cross-contaminated, while the I region was heavily contaminated by the LL material (Figure 5B2). The I molecules contained some heavy label, but not an entire heavy strand. Their radioactivity must be mostly due to the H strand because (i) H strands contained 62% of radioactivity before transfer of density labelled cells to the light medium and the total amount of radioactivity present in the HL peak and I region was ~60%; and (ii) the specific activity of the L strands, which were synthesized continuously, was much lower than that of H strands (compare the amounts of
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Analysis of density labelled plasmids. Hfr3.OSOΔ64 cells containing pHV857 were radioactively and density labelled by growth in the presence of [³H]thymidine and deoxybromouridine. Plasmid DNA was extracted and analysed by CsCl density centrifugation. HH, HL and LL denote double-stranded molecules with two, one and no dense strands. I refers to molecules with less than one entire dense strand. Fractions of the gradient were dialysed and analysed by Southern hybridization using pHV857 as probe. Positions of parental supercoiled monomers (1), nicked monomers and dimers (2) and oligomers (3) are indicated. R and S denote the positions of the recombinant supercoiled monomers and of the single-stranded DNA (parental and recombinant), respectively. Faint bands migrating more quickly than parental and recombinant monomers, detected in some samples (A and B) between HL and LL peaks, are due to traces of DNA denatured during extraction, as deduced from (i) their position relative to appropriate markers; (ii) their resistance to restriction enzymes and (iii) their increase in amount when a higher volume of alkaline solution was used during cell lysis (not shown). (A) Cells grown in heavy medium only. Fractions 4–10, 11–20 and 21–26 were pooled for the second cycle of centrifugation (A1–3, respectively). (B) Cells grown in heavy medium then in light medium uninfected by phage f1. Fractions 9–17, 18–21 and 22–27 were pooled for the second cycle of centrifugation (B1–3, respectively). (C) Cells grown in heavy medium then in light medium infected by phage f1. Fractions 9–14, 15–19 and 20–23 were pooled for the second cycle of centrifugation (C1–3, respectively). The average recovery of the radioactive material in the second cycle of centrifugation was 55% and varied between 35 and 70%.</figcaption></figure>
<table><caption>**Table I.** Proportion of recombinant plasmids among molecules of different density<sup>a</sup></caption><thead><tr><th rowspan="2">Type of molecule</th><th colspan="3">Proportion of recombinants (Ap<sup>R</sup>/Tc<sup>R</sup> transformants × 10<sup>4</sup>)</th></tr><tr><th>Culture medium<br>Heavy</th><th>Light without f1</th><th>Light with f1</th></tr></thead><tbody><tr><td>HH</td><td>2.3 ± 1.4 (&lt;)</td><td>np<sup>b</sup></td><td>np</td></tr><tr><td>HL</td><td>2.1 ± 1.9 (&lt;)</td><td>3.1 ± 0.7</td><td>6.5 ± 5.3</td></tr><tr><td>I (ds)<sup>c</sup></td><td>np</td><td>6.2 ± 4.3 (&lt;)</td><td>850</td></tr><tr><td>I (ss)<sup>c</sup></td><td>np</td><td>np</td><td>700</td></tr><tr><td>LL</td><td>3.0 ± 2.0</td><td>1.0 ± 0.5</td><td>460 ± 110</td></tr></tbody></table>

<sup>a</sup>Labelling was with BUDR. DNA was prepared essentially as shown in Figure 5. Electrotransformation was used for double-stranded DNA (with efficiency of 5×10<sup>8</sup> to 1×10<sup>9</sup> transformants/µg DNA) and CaCl<sub>2</sub>-dependent transformation (with efficiency of 5×10<sup>6</sup> to 1×10<sup>7</sup> transformants/µg DNA) for single-stranded DNA, which does not electrotransform efficiently. Five series of independent transformations were performed for HL molecules prepared from the cells grown in the presence of f1, one or two series for the other molecules. The sign (<) indicates that no Ap<sup>R</sup> transformant was obtained in some experiments.

<sup>b</sup>Not present.

<sup>c</sup>Prior to transformation DNA was treated with S1 nuclease to obtain the double-stranded (ds) sample and with *HphI* restriction endonuclease to obtain the single-stranded (ss) sample.

radioactivity and DNA in HL and LL peaks; Figure 5B). We suggest that the I molecules were formed by homologous recombination between the HL and LL molecules. Further arguments in favour of this hypothesis are presented in the following paragraph.

The infected cells contained HL, LL and I molecules, carrying ~25, ~30 and ~45% of radioactivity, respectively (Figure 5C). Southern hybridization indicated that LL and HL molecules were double stranded, while the I molecules were predominantly single stranded. Control experiments (density centrifugation of single-stranded DNA standards; measurements of the radioactivity of the relevant portions of the gel) established that the single-stranded molecules were light and carried very little radioactivity (not shown). Double-stranded I molecules were therefore radioactive and must have contained heavy label, but in only about one third of a strand, judging by the position of the I peak in the gradient (Figure 5C).

In addition to the arguments developed above, two further observations support the notion that double-stranded I molecules form by homologous recombination between molecules of different density: (i) their amount was higher in infected than in uninfected cells (compare the radioactivity profile of Figure 5B with C) and it is known that rolling circle replication stimulates homologous recombination (Noirot *et al.*, 1987); and (ii) chromosomal DNA of intermediate density was not detected (not shown), which rules out re-incorporation of BUDR into otherwise light DNA upon infection. As expected, most of the double-stranded DNA was light and, irrespective of its density, parental. However, the recombinant DNA is clearly visible at the LL and I positions, which shows that the density labelling protocol did not prevent stimulation of transposon excision. A similar proportion of HL recombinant DNA would not be detected because of the relatively low amount of the parental HL DNA. It should be noted that single-stranded

H DNA was not detected (it would be present in the first fractions of the gradient) and that the total radioactivity in the HL and I molecules was somewhat lower (55%) than that found in the H strand before transfer to light medium (62%, see above). In control experiments, we have not been able to prevent a drastic loss of single-stranded H DNA during centrifugation, by adsorption on centrifugation tubes, despite coating the tubes before use by siliconization and/or incubation with large amounts of single-stranded DNA (not shown). HL, I and LL molecules were purified further by centrifugation (Figure 5C1-3). We estimate by Southern analysis that the top HL and light-side LL fractions were essentially pure.

To determine the proportion of recombinant plasmids among molecules of different type (HH, HL, LL and I), the fractions of highest purity from each of the relevant repurified peaks were used to transform *E. coli* cells. Different levels of nicking observed with different fractions (Figure 5) should not affect the comparisons, provided that the parental and recombinant molecules are nicked to the same extent in each sample, which is very likely. The results are presented in Table I. As expected, the proportion of recombinants was low among molecules prepared from the uninfected cells, irrespective of the culture medium (heavy or light without f1) and the type of molecule (HH, HL or LL). It was much higher among LL and I (ds and ss) molecules prepared from the infected cells. Most importantly, the proportion of recombinants among the HL molecules from the infected cells was not significantly higher than among the molecules from the uninfected cells. This shows that recombinant molecules do not contain an entire DNA strand donated by parental molecules upon transposon excision.

To verify that the incorporated BUDR does not affect the fate of DNA during recombination, the cells were density labelled with heavy isotopes (¹³C and ¹⁵N), recombination was induced by phage infection and the relevant molecular types purified by centrifugation (three cycles of purification were used rather than two, because the separation of different molecular forms was less favourable than for BUDR labelling; not shown). In a representative experiment the proportion of recombinant plasmids prepared from infected cells was 6.3×10⁻⁴ and 370×10⁻⁴ among the HL and LL molecules, respectively, close to values observed with the BUDR-containing DNA (Table I).

## Discussion

Break and join recombination transfers DNA from the parental to the recombinant molecule, whereas copy-choice recombination does not (Meselson and Weigle, 1961). The two types of process can therefore be unambiguously distinguished by following the fate of DNA during recombination. This is possible if the parental molecules can be labelled under conditions where recombination is low and can be stimulated to a sufficient level at the end of the labelling period. It was suggested that transposon excision from replicons which undergo rolling circle replication via a single-stranded DNA intermediate is much more efficient than from those that do not (Berg *et al.*, 1983; Jannière *et al.*, 1990). We therefore used plasmids which replicate as theta molecules, but in which rolling circle replication can be induced, and showed that induction of rolling circle
replication greatly stimulates nearly precise transposon excision. The plasmids were density labelled, using BUdR or heavy isotopes ($^{13}$C and $^{15}$N), under conditions of low excision frequency and excision was stimulated in a medium free of the density label. The newly formed recombinant plasmids never contained an entire density labelled DNA strand, which rules out a simple break and join recombination process. A process which would break and transfer only about one third of a DNA strand from a parental to a

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Nearly precise transposon excision by copy-choice recombination. Thick and thin lines represent pre-existing and newly synthesized DNA strands. Inverted repeats of the transposon are delimited by a vertical line and a filled arrowhead. Direct repeats are represented by empty triangles. (1) Single-stranded DNA is generated by rolling circle replication and a hairpin structure is formed. (2) Complementary strand synthesis has penetrated into the hairpin structure past the direct repeats. (3) The tip of the newly synthesized strand is extruded from the hairpin and the direct repeat it carries is annealed with the non-replicated homologue. (4) The tip has been trimmed and used as a primer for further replication. (5) A double-stranded recombinant is formed by another round of DNA replication.</figcaption></figure>

recombinant molecule during transposon excision could not be ruled out formally because of intermediate density plasmids formed in our experiments. These plasmids could either be a direct and obligatory product of transposon excision or be formed regardless of transposon excision. The first possibility is unlikely because the proportion of recombinants was not significantly higher among the intermediate density plasmids than among fully light plasmids, while the former should have contained a very high proportion of recombinants if they arose as a direct product of transposon excision. Furthermore, it is not easy to conceive an excision process which implies re-synthesis of two thirds of the recombinant plasmid strand and which is stimulated by rolling circle replication. In contrast, the second possibility is much more likely, because several observations suggest that intermediate density plasmids were formed by homologous recombination between molecules containing one and no dense strands. If different light molecules (parental and recombinant) recombined with similar efficiencies with heavy/light molecules (which are parental), the proportions of recombinants among the light and intermediate density molecules should not be very different, as observed. We therefore consider it very probable that transposon excision occurs by copy-choice recombination, as represented in Figure 6. Similar models for recombination between short direct repeats have been proposed previously (Egner and Berg, 1981; Albertini et al., 1982; Weston-Hafer and Berg, 1989, 1991).

The experiments reported here raise two questions. One concerns the enzymes involved in transposon excision. Two recent reports suggest that copy-choice recombination might take place on the lagging strand template (Trinh and Sinden, 1991; Gordenin et al., 1992). In agreement with this observation the recombinant plasmids were detected earlier in double-stranded than in single-stranded form in our experiments (Figure 4). Because leading and lagging strand synthesis are uncoupled during rolling circle replication a

<table><caption>**Table II.** Bacterial strains, plasmids and phages</caption><thead><tr><th></th><th>Genotype</th><th>Reference</th></tr></thead><tbody><tr><td><strong>Strains</strong></td><td></td><td></td></tr><tr><td>Hfr3.OSOΔ64</td><td>Hfr3 thi lac thyAΔ64</td><td>Alikhanian et al. (1966)</td></tr><tr><td>JJC40</td><td>thi-1 proA2 his-4 argE3 lacY1 galK2 ara-14 xyl-15 mtl-1 tsx-33 rpsL31 supE44 hsdR</td><td>Bierne et al. (1991)</td></tr><tr><td>JJC41</td><td>JJC40 (F'<i>lac</i><sup>I</sup> ZΔM15 pro<sup>+</sup>)</td><td>This work</td></tr><tr><td>JJC42</td><td>as JJC40 but <i>rep-3 pro</i><sup>+</sup></td><td>Bierne et al. (1991)</td></tr><tr><td>JJC41 <i>leu</i><sup>+</sup></td><td>Leu<sup>+</sup> revertant of JJC41</td><td>This work</td></tr><tr><td><strong>Plasmids</strong></td><td></td><td></td></tr><tr><td>pHV60</td><td>pBR322 carrying Cm<sup>R</sup> gene of pC194</td><td>Michel et al. (1982)</td></tr><tr><td>pHV60oriM13</td><td>TaqI fragment 5680-6039 of M13 inserted into <i>Clal</i> site of pHV60</td><td>This work</td></tr><tr><td>pHV857</td><td>pHV60oriM13 with miniTn10-Km (Way et al., 1984) inserted into Ap<sup>R</sup> gene</td><td>This work</td></tr><tr><td>pHV857gpV</td><td>EcoRI-BamHI fragment of pLV (Fulford and Model, 1988a,b) in <i>PvuII</i> site of pHV857</td><td>This work</td></tr><tr><td>pSCgpII</td><td>Spectinomycin-resistant derivative of pSC101 carrying gene II of <i>f1</i> controlled by <i>lacUV5</i> promoter</td><td>Uzest et al. (1991)</td></tr><tr><td>pAClacI<sup>I</sup></td><td>Replication region and Cm<sup>R</sup> gene of pACYC184 (Chang and Cohen, 1978) linked to the <i>lacI</i><sup>I</sup> gene of pS01000 (Oehler et al., 1990)</td><td>This work</td></tr><tr><td><strong>Phages</strong></td><td></td><td></td></tr><tr><td>M13</td><td></td><td>van Wezenbeek et al. (1980)</td></tr><tr><td><i>f1</i></td><td></td><td>Beck and Zink (1981)</td></tr><tr><td>λ1105</td><td></td><td>Way et al. (1984)</td></tr></tbody></table>
different replication complex is probably used in the two
cases. It would be interesting to know whether the presence
of certain components of the complex might affect the
frequency of the slippage. For instance, the M13 gene V
product, which is an SSB protein, was required for
stimulation of excision and could not be replaced by the host
SSB. The role of this protein during M13 infection is to
favour accumulation of single-stranded DNA by preventing
initiation of the complementary strand synthesis from the
phage (-) origin (Model and Russel, 1988). However, the
plasmids used here lack this origin and initiation of their
complementary strand synthesis is presumably mediated by
a primosome assembled at the *rriA* site of pBR322 (Zipursky
and Marians, 1980). The effect of the gene V protein on
slippage of DNA synthesis initiated at primosome-generated
primers is not known. It should, however, be noticed that
recombination between short direct repeats might also occur
during leading strand synthesis, as suggested by a recent
study (Weston-Hafer and Berg, 1991).

The second question is how general the copy-choice
mechanism might be. The model we used, the nearly precise
transposon excision, occurs by recombination between the
direct repeats embedded within inverted repeats, and requires
single-strand DNA synthesis. Clearly, many illegitimate
recombination events involve direct repeats which are not
within, or even close to, inverted repeats. Because the
presence of inverted repeats stimulates recombination
between direct repeats (Foster *et al.*, 1981; Peeters *et al.*,
1988; Weston-Hafer and Berg, 1991) it is conceivable that
it modifies the recombination mechanism. However, the
stimulation could be due simply to the formation of hairpin
structures by annealing of the inverted repeats, which should
bring the direct repeats closer together, because it is known
that recombination frequency between direct repeats
increases greatly when their distance decreases (Scearce
*et al.*, 1991; Chedin *et al.*, 1994). Also, hairpin structures
are known to arrest the replication forks (LaDuca *et al.*,
1983), and therefore more time for slippage of the growing
tip of a DNA strand might be provided. Thus, the copy-
choice recombination might also occur in the absence of the
inverted repeats, although with lower frequency.

With regard to the requirement for single-stranded DNA,
the replicons which use rolling circle (or rolling hairpin)
replication, such as many bacterial episomes (phages and
plasmids; Denhardt *et al.*, 1978; Willets and Wilkins, 1984;
Gruss and Ehrlich, 1989) and possibly also certain plant and
animal viruses (Ilyina and Koonin, 1992), accumulate this
this DNA form. In replicons which use thetaph replication
(chromosomes and many episomes) the template of the
discontinuously synthesized strand is expected to exist
periodically in single-stranded form (Kornberg and Baker,
1992). Therefore, the substrate for copy-choice
recombination should be available in any genome, although
its levels are certainly not the same in all of them.
Furthermore, it should be noted that, formally, copy-choice
recombination requires only the short homologous sequences
to be single stranded and the remainder of the molecule may
remain double stranded. These considerations support the
notion that many events reported previously of illegitimate
recombination involving short homologous sequences might
be due to a copy-choice process similar to that detected in
the model system used here. The effect of various parameters
which affect the frequency of the process on the mechanism

of recombination should, however, be much better
understood before the conclusions reached in this work can
be generalized.

**Materials and methods**

Bacterial strains, plasmids and growth conditions

Bacterial strains and plasmids are listed in Table II. The bacteriophage λ105 was used to insert the miniTn10-Km transposon on pHV60-oriM13, as described (Way *et al.*, 1984). Standard rich and minimal media (LBT, which is LB supplemented with thymine to 50 mg/l and M63, respectively; Miller, 1972) were used for cell growth. For density labelling experiments with BUdR, M63-B was used, which is M63 supplemented with 4 g/l glucose, 4 g/l hydrolysed casein, 100 mg/l tryptophan, 50 mg/l deoxyguanosine, 66 mg/l uridine and 10 mg/l vitamin B1. For density labelling with heavy isotopes, M63-A was used, corresponding to M63 without ammonium sulfate. Antibiotics were used at the following concentrations: ampicillin 100 µg/ml, tetracycline 7.5 or 15.0 µg/ml, kanamycin 30 µg/ml, spectinomycin 30 µg/ml and chloramphenicol 25 µg/ml. Transformation was carried out by CaCl₂ treatment or electroporation, as described (Dagert and Ehrlich, 1979; Calvin and Hanawalt, 1988).

Kinetics of recombination induced by f1 infection

Cells carrying the parental plasmid were grown overnight in LBT
supplemented with kanamycin. The culture was diluted 250-fold and cells
were grown in LBT supplemented with tetracycline (15 µg/ml) to an OD<sub>650</sub>
of 0.5. It was then divided in two; one part was infected by f1 and the
other was not. Infection was synchronized by incubating the cells,
concentrated by centrifugation to 10⁹ cells/ml, with 5×10¹¹ phages/ml for
10 min at 37°C. More than 90% of the cells were infected by this time,
as shown in control experiments. The infected cells were diluted 100-fold
into fresh prewarmed LBT medium and grown at 37°C to allow
recombination.

Kinetics of recombination induced by production of phage
replication proteins

Cells carrying pHV857gpV, pSCgpII and pAClaI⁹ were grown overnight in LBT medium supplemented with tetracycline (15 µg/ml), spectinomycin and chloramphenicol. They were diluted 1000-fold in LBT medium and grown until OD₆₅₀ = 0.05–0.10. The culture was divided in two; one part was used as a control and in the other part the phage replication genes were induced by addition of 1 mM IPTG.

DNA preparation and analysis

Plasmid DNA was extracted from *E. coli* by a scaled-up method of Birnboim and Doly (1979), followed by purification by a CsCl density gradient. Total DNA was extracted from *E. coli* as described by te Riele *et al.* (1986a). Single-stranded DNA was detected in total DNA extracts by hybridization (te Riele *et al.*, 1986b). For cloning and restriction analysis, enzymes from BRL, Boehringer and New England Biolabs were used as recommended by the supplier. Analyses of the miniTn10-Km insertion sites and recombination events were carried out by sequencing with Sequenase on double-stranded DNA template with synthetic primers. BUdR-labelled DNA was analysed by centrifugation in CsCl solution adjusted to a density of 1.7422 g/ml in a VTi 65.1 rotor (Beckman) for 25 h at 43 000 r.p.m. For heavy isotope-labelled DNA the density was 1.7420 g/ml and the centrifugation speed was 43 000 r.p.m. for the primary separation of labelled molecules and 32 000 r.p.m. for the two subsequent purification steps. Fractions were collected from the bottom of pierced tubes and extensively dialysed against buffer, containing 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA, before analysis by hybridization or transformation.

Density labelling

*Bromodeoxyuridine.* Cells from 1 ml Hfr3.OSOΔ64 culture grown overnight in LBT were collected by centrifugation, washed twice in M63 and transferred to 120 ml M63-B supplemented with tetracycline, 2 mg/l thymidine and 5 µCi/ml [6-³H]thymidine (Amersham). Cells were grown for 4 h at 37°C (approximately four generations) to label their DNA. They were collected by centrifugation, washed twice in M63, transferred to 120 ml prewarmed M63-B supplemented with 10 mg/l bromodeoxyuridine and 6 µCi/ml [6-³H]thymidine and grown for 1 h at 37°C. 20 ml of the culture were used for DNA extraction, 50 ml for M13 phage infection and 50 ml for uninfected control. Cells were collected from the last two samples by centrifugation, inoculated in 500 ml LBT and grown at 37°C for 3 h (four to five generations).
**Heavy isotopes.** Cells from 5 ml Hfr3.**OSOΔ64** culture grown overnight in M63 supplemented with kanamycin, 50 mg/l thymidine and 1.6 g/l glucose were collected by centrifugation, washed twice in M63-A and inoculated in 125 ml of M63-A supplemented with 2 mg/l thymidine, 5 µCi/ml [6-³H]thymidine, 400 mg/l [¹³C]d-glucose (Interchim) and 2 mM [¹⁵N]ammonium sulfate (Sigma). They were grown for 5 h at 37°C (approximately four generations). 25 ml of the culture were used for DNA extraction, 50 ml for M13 phage infection and 50 ml for uninfected control. The last two samples were treated as in the case of BUDR labelling.

## Acknowledgements

We thank C.Anagnastopoulos for help in preparing the manuscript and F.Haimet and B.Nicolas for artwork. This work was supported, in part, by the EEC grant BIOT-CT91-0268.

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Received on September 10, 1993; revised on March 2, 1994