Intramolecular recombination of linear DNA catalyzed by the Escherichia coli RecE recombination system

J. Mol. Riol. (1985) 186, 515-525

Intramolecular Recombination of Linear DNA Catalyzed by the Escherichia coli RecE Recombination System Lorraine S. Symingtonl~ 2, Paul Morrison’

and Rochard Kolodner1*2

‘Laborato r y of Molecular Genetics Dana-Farber Cancer Institute 44 Binney Street, Boston, MA 02115, U.S.A. 2Department of Biological Chemistry Harvard Medical School Boston, MA 02115, U.S.A. (Received

20 February

1985, and in revised form

16 July

1985)

Transformation of different Escherichia coli strains by linear dimers of pBR322 containing different tet alleles was investigated. Linear dimers transformed wild-type strains 0.1 to lo/ as efficiently as circular dimers. In contrast, linear dimers transformed recBrecCsbcA strains, where the RecE recombination system is functional, as efficiently as circular dimers. The transformants contained plasmids that had a single recombinant monomer genotype, indicating that transformation was mediated by a recombination-dependent cyclization reaction. Altering the position of the double-strand break changed the frequency of recovering different recombination products, but had no effect on the frequency of transformation. Both the frequency of transformation and the production of Tc’ recombinants were decreased by recE mutations, while recA and recF mutations were slightly stimulatory (twofold). Several recombination models consistent with these result’s are presented.

1. Introduction

Conjugation-mediated recombination events in recBrecCsbcA E. coli strains require at least the products of the recA, recF, and recJ genes, and in addition, require the product of the recE gene (Clark, 1980; Gillen et al., 1981; Lovett & Clark, 1984). Recombination in recBrecCsbcA E. coli strains has been called the RecE recombination pathway, although in this communication it will be called the RecE recombination system because sbcA mutations have multiple effects on recombination. Genetic analysis of plasmid recombination has demonstrated that the frequency of plasmid recombination events in wild-type E. coli strains is decreased by recA, recF, recJ, rec0, ssb and topA mutations and is increased by mutations that derepress the recA/lexA regulon (Fishel et al., 1981; Fishel & Kolodner, 1984; James et al., 1982; Kolodner et al., 1985; Laban & Cohen, 1981; Cohen & Laban, 1983). Thus, plasmid recombination in wild-type E. coli strains appears to be catalyzed by a pathway that is similar to, but not identical with, the RecF pathway. In recBrecCsbcA strains, the frequency of plasmid recombination events is decreased by recE mutations while recA and recF

Genetic analysis has demonstrated that there are multiple pathways or mechanisms for homologous recombination in Escherichia coli. The isolation of mutations that decrease the frequency of recombination events after mating in wild-type E. coli strains has led to the identification of three genes, recA, recB, and recC, that define the RecBC recombination pathway (Clark & Margulies, 1965; Emmerson & Howard-Flanders, 1967; Willetts et al., 1969). The isolation of two mutations, sbcA and sbcB. that suppress the recombination defect present in E. coli strains containing both the recB and reck mutations led to the discovery of alternative pathways for recombination (Barbour et al., 1970; Templin et al., 1972). Genetic analysis of conjugation-mediated recombination in recRrvcCsbcR E. coli strains has demonstrated that recombination in these strains requires the products of the recA, recF, recJ, recN, rec0 and ruv genes (Lloyd et al., 1983, 1984; Clark, 1980; Horii & Clark, 1973; Kolodner et al., 1985; Lovett & Clark, 1984). Recombinat’ion in recBrecCsbcB E. coli strains has been called the RecF recombination pathway. 515

0

1985

Academic

Press

Inc.

(London)

Ltd.

516

L. S. Symington,

P. Morrison

mutations have no effect (Fishel et al., 1981; James et al.. 1982; Joseph, 1983; Laban & Cohen, 1981; Cohen & Laban, 1983). The RecE recombination system has been of major interest to us because it catalyzes plasmid recombination events at least 20 times more frequently than the RecF-like recombination pathway (Fishel et al., 1981; James et al., 1982; Laban & Cohen, 1981). sbcA mutations induce the synthesis of exonuclease VIII, the recE gene product (Gillen et aE., 1981; Lloyd & Barbour, 1974: Low, 1973; Kushner et aZ., 1974). Detailed enzymological studies of exonuclease VIII have shown that exonuclease VITI is similar to 2 exonuclease, although the two enzymes differ greatly in molecular weight (Joseph & Kolodner, 1983a,b). Exonuclease VIII, like 1. exonuclease, degrades linear duplex DXA and does not appear to be able to degrade covalently closed, nicked or circular substrates even though it gapped participates in recombination events between circular DNA molecules (Joseph & Kolodner, 1983a,b; Carter & Radding, 1971: Radding & Carter, 1971). This suggests that either exonuclease VTTI has other activities (functions in conjunction with other enzymes, acts on a recombination intermediate) or that plasmid recombination might involve a linear substrate or intermediate (Joseph, 1983). In this communication we report the results of experiments designed to determine whether the Reck system will act on linear DNA molecules.

2. Methods (a) Bacterial

strains

All of the bacterial strains used in this studv were isogenic derivatives of the E. coli K12 strain ABl l”57 Fthr-1, leuB6, thi-1, lacYI, galK2, ara-14, ~~1-5, mtl-1, proA2, rpsL31, tsx-33, his-4, argE3, supE44, 1”. I.-. These strains were JC10287 A (recA-srlR)304, JC5519 recB21recC22, JC9239 recF143, JC13031 recJ153, JC8679 recB21recC22sbcA23, JC9604 recA56recB21recC22sbcA23, JC9610 recF143recB21recC22sbcA23, and JC869 1 recE159recB21recC22sbcA23. These strains were kindly provided by Dr A. J. Clark. Plasmids were maintained and propagated in JC10287. All strains were grown at, 37°C in L-broth (1% (w/v) tryptone. 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl) or on T-broth (1% (w/v) tryptone, 0.59; (w/v) NaCl) plates or L-broth plates containing 1.50/b (w/v) agar. When appropriate, media were supplemented with ampicillin at 50pg/ml and/or tetracycline at 25 pg/ml. (b) Preparation

and analysis of plasmid

DNA

Large-scale preparations of plssmid DNA were purified by two cycles of CsCl/ethidium bromide density gradient centrifugation, essentially as described (Kolodner, 1980: James et al., 1982). This DNA was used for all plasmid constructions and transformation experiments. Smallscale plasmid preparations were prepared from individual transformants as described by Holmes & Quigley (1981), except that sometimes the DNA was further purified by extraction with phenol and precipitation with ethanol. Restriction endonucleases were purchased from New England Biolabs (Beverly. Mass.) and used according to

and R. Kolodner

the instructions supplied by the manufacturer. unless otherwise specified. Electrophoresis in agarose slab gels containing ethidium bromide was carried out as described (Fishel et al., 1981: James et al., 1982). (c) Recombination

substrates

The plasmid pRDK41 has been described (Doherty et al., 1983) and is a circular dimer of pBR322 that contains one copy each of the tet-10 and tet-14 mutant alleles (Fig. 1). To insert an XbaI linker (d(C-T-C-T-A-G-A-G). (:ollaborative Research. Waltham, Mass.), individually at each of the two BamHI sites in pRDK41, 5 pg pRDK41 DNA were digest’ed with BamHT for 1 h at 37°C in a 200 ~1 reaction mixture that contained 150 enzyme units of BamHI/ml and 9 pg ethidium bromide/ml. EDTA (pH 8.0) was then added t,o a concentration of 10 mM and the DNA was purified by extraction with phenol and precipitation with ethanol. The resulting DNA, which contained approximately 509, linear dimers, was incubated wit,h reverse transcriptase to fill in the BamHT sticky ends and XbaI linkers were added on to the blunt ends exactly as described (James et al.. 1982: Doherty rt al., 1983). The DNA was then digested with 500 units of XbaI, fractionated by elect,rophoresis on an agarose gel and the linear dimer band was excised from the gel and the linear dimer DNA was purified as described (Weislander. 1979). The linear dimer DXA was then cvclized with phage T4 DNA ligase and used to transform either E. coli JC10287 or E. coli RK1400 (Doherty of al.. 1983; Symington et al., 1983). Plasmid DNA preparations were prepared from individual transformants and analyzed by digestion with either BamHI, XbaI or XhoI. or by double digestion with XhoT+XhaI t,o identif! derivatives of pRDK41 that, contained an XbaI linker inserted into one of the two Ban&HI sites such that t,he BamHI site had been regenerated. The plasmid pRDK68 has an XbaT linker inserted in the BamHI site closest. to the tet-20 mutation and pRDK69 has an XbaI linker inserted in the BamHI site closest to the tet-2-l mut’ation (Fig. 1). XbaI linkers were inserted into the PzwII sites of pRDK41 using essentially the same procedure except that the restriction endonuclease digestion reaction contained PwII at 75 enzyme units/ml and ethidium bromide at 25 pg/ml. and t,he incubation with reverse transcriptase was omitted since PauII makes blunt ends. The plasmid pRDK70 has an XbaI linker inserted at the PvuII site t~hat is closest to the tet-10 allele and the plasmid pRDK71 has an XbaI linker inserted at the PvuIT site that is closest to the tet-14 allele (Fig. 1). Linear dimer recombination substrat’es were prepared from pRDK68, pRDK69, pRDK70, and pRDK71 DNA by digestion with XbaI, followed by purification of the DN\;s by extraction with phenol and precipitation with rt,hanol. Linear dimers of pR’DK41 were prepared by pa,rtial digestion with BamHI in t)he presence of rthidium bromide, using the digestion conditions described ahovr. Thta DNA was then fractionated by electrophoresis in an agarose slab gel followed by excision of the linear dimer band from the gel and purification of the linear dimcr D?I;A essent,ially as described (Weislander. 1979). Linear dimers of pRDK41 were also prepared by partial digestion of pRDK41 D8A wit,h P8tI using essential]) the same procedure except that 5 pg pRDK41 DS,4 were digested with 20 units of PstI at 30°C for I5 min in a 100 ~1 reaction containing 10 mM Tris. HC‘I (pH 7.4). 10 mwMgC1,. 1 mm-dithiothreitol and 50 m.n-ll’aCl. To test the substrate preparations for purity, each linear dimer preparat,ion was analyzed by electrophoresis on an

Intramolecular

Recombination

of Linear

517

DNA

Table 1 Transformation

of Escherichia

coli by pRDK41

and pRDK41jBamHI

Ap’ transformants Strain

tested

ABll57 ,J(‘IO287 ,JC:,55 19 5(‘9239 . s ,JCl3031 J(38679 ,J(!9604 JC8691 JC9610

pRDK41

Transformation a Total number of Ap’ transformants

used in our studies, visible when 0.5 pg of

that our substrate were >98 to 99% pure linear dimers. (d) Recombination

tests

Competent E. coli cells were prepared and transformed essentially as described with each transformation mix containing 0.05 t,o 02 pg of plasmid DPJA (Cohen et al., 1972; Wensink ef al., 1974). Transformations of a given strain were carried out in pairs and contained either circular dimer DPU’A or an equal amount of the indicated linear derivative of the rircular dimer. Transformed cells were plated on L-broth plates containing ampicillin, or ampicillin and tetracycline to determine the number of total transformants and the number of transformants containing tetracyrline-resistant (Tc’)~ recombinant molecules. respectively. The efficiency of transformation (L/C) by linear DKA was expressed as the ratio of the number of transformants obtained with linear dimer DKA (L) divided by the number of transformants obtained with circular dimer DiTA (C) for a pair of transformations.

3. Results (a) Tmnsformation

by circular

and linear

dimers

We hare examined the ability of linear dimers to transform a variety of E. coli strains. The results presented in Table 1 show that linear dimers transformed wild-t’ype E. coli to ampicillinresistance (Ap’) less than lqio as efficiently (L/C’) as the circular dimer pRDK41 did. Mutations in the rrrA, recBrec(‘. recF or recJ genes had little effect on the eficaiency of t*ransformation by linear dimers. In contrast to the result’s obtained with wild-type E. coli, linear pRDK41 DNA t’ransformed E. coli JC8679 rwBrecCsbcA almost as efficiently as circular pRDK41 DNA. ret/l and recF mutations in this genetic background had litHe effect on the transformat,ion efficiency (L/C) of linear pRDK41.

t Abbreviations

ampicillin-resistant;

To’. tetracycline-resistant: kb. IO3 base-pairs.

used:

x x x x x x x x x

IO6 lo6 lo5 lo5 lo6 lo5 103 lo5 10’

tests were carried out as described under Methods. of Ap’ transformants obtained with pRDKBl/BamHI obtained with pRDK41.

agarove gel. In all of the preparations only the linear dimer band was DEA was analyzed, indicating

preparations

1.1 1.6 2.6 8.3 1.5 1.8 6.3 1.3 1.6

ArecA.303 recBZlrecC’22 reeF143 rec~f,S.? recBZlrecC22sbcA23 rrcB2lrecC22sbcA23reeA56 vecBZlrecC22sbcA23recEl59 recB2lrwC22sbcA23recF143

Ap’,

per ml with pRDK41/BamHI 4.4 x 103 5x lo3 1.3 x lo3 5 x lo3 1.2 x lo4 1.4x lo5 4.2 x lo3 7.2 x lo3 7x lo4

divided

L/c’” 0.004 0.0031 0.005 0.006 0.008 1 0.78 0.68 0.056 0.44

by the total

number

while a recE mutation reduced the transformation efficiency of linear pRDK41 DNA by 14.fold. We have also monitored the formation of recombinant Tc’ plasmids after transforming different E. coli strains with either circular or linear dimers (Table 2). In E. coli ABll.57, the frequency of obtaining Tc’ recombinants was increased 50-fold when the dimeric recombination substrate was linearized and there was a fivefold decrease in the actual number of Tc’ transformants obtained. The data presented in Table 2 indicated that the recA and recJ mutations decreased the production of Tc’ recombinants with linear pRDK41 DNA by 14-fold and l&fold, respectively, while the other mutations tested had little, if any, effect. The effects of these mutations on recombination of circular dimers are consistent with results presented elsewhere and are presented here only as controls (Doherty et al., 1983; Fishel et al., 1981; James et al., 1982; Kolodner et al., 1985; Laban & Cohen, 1981; Cohen & Laban, 1983). In E. coli tJC8679 recB2lrecC22sbsA23, recombination of linear pRDK41 to produce Tc’ recombinants was sixfold higher than recombination of circular pRDK41. In a ret BrecCsbcA background, recA and recF mutations increased the frequency of recovery of Tc’ recombinant’s twofold while the recE mutation in a recBrecCsbcA background decreased the production of Tc’ recombinants sixfold. These results are similar to earlier findings on the recombination of circular plasmids in ret BrecCsbcA E. coli strains (Fishel et al., 1981; James et al., 1982; Joseph, 1983; Laban & Cohen, 1981: Cohen & Laban. 1983). To examine the effect of the position of t,he double-strand break and the type of end present on transformation with linear dimers, transformation with six different linear dimer substrates was examined. The results (Table 3) indicate that changing the position of the double-strand break or the type of end (5’ or 3’ single-stranded overhang) had little effect on the frequency of transformation of E. coli JC8679 recBrecCsbcA by linear dimeric DXA. The position of the double-strand break

518

L. S. Symington,

P. Morrison

and R. Kolodner

Table 2 Frequency

of obtaining

recomb&ants

with pRDK41

Tc’ transformants” Strain

tested

AS1 157 JClO287 JC.5519 JC9239 JCl3031 JC8679 JC9604 JC8691 JC9610

pRDK41 6.4 1.3 7.8 1.2 7x 2.5 3.3 1x 1x

ArecA304 recB2lrecC22 reeFI recJ15.3 recB21recC22sbcA23 reeB21recCZZs~A23~ecA56 recB21recC22sbcA23recE159 recB2lrecC22sbcA23recF143

x 10’ x IO2 x IO2 x lo2 10’ x 10’ x 10’ lo2 103

and pRDK4lIBamHI

per ml with

Recombination

pRDK41/BamHI 1.2 < 10 1x 8x 2x 1.2 5.9 1x 1.1

pRDK4

x lo2

frequency

1

pRDK41/BamHI

0.058 O-0081 0.3 0.014 0.0046 1.4 0.53 0.08 0.64

102 10’ 10’ x lo4 x lo2 lo2 x 104

2.8 <0.2 0.77 1.6 0.16 8.2 14 1.4 16

a The transformation mixes obtained in the experiment described in Table 1 were plated on plates containing tetracycline to determine the number of Te’ transformants present. b The number of Ap’ Tc’ transformants divided by the number of Ap’ transformants obtained in the same experiment given in Table 1

relative to the position of the mutant tet alleles had a large effect on both the production of Tc’ recombinants in E. coli JC8679 (Table 3) and on the frequency of recovery of certain other recombinant species (Table 4). All of the linear dimers tested transformed E. coli AB1157 inefficiently (Table 3). (b) Structure

of the recombination

products

To gain some insight into the mechanism of recombination of linear dimer DNA after transformation into E. coli JC8679 recBrecCsbcA, the structure of the plasmid DNA obtained from individual transformants was determined by restriction mapping. The analysis of the structure of some of these plasmids is presented in Figure 2 and restriction maps of all of the plasmids observed are presented in Figures 1 and 3. All of the Tc’ recombinants that arose by transformation of E. coli JC8679 recBrecCsbcA with pRDK41/BamHI contained a series of circular oligomers (Fig. 2(a), lane 4). These circular oligomers were resistant to digestion with XhoI (Fig. 2(a), lane 5) and yielded 4.36 kb linear molecules on digestion with

L/C’ Linear

substrate

pRDK/BamHI pRDK41/PstI pRDK6S/XbaI pRDK69lXbaI pRDK70/XbaI pRDK’Il/XbaI

DNA

obtained

with

ampicillin

and

using the data

XhoI+PstI (Fig. 2(a), lane 6). These data are consistent with all of the circular oligomers having wild-type pBR322 (Fig. 3) as a repeating monomer unit. All of the Ap’ transformants that arose by transformation of E. coli JC8679 recBrecCsbcA with pRDK4lIBamHI also contained a series of circular oligomers (Fig. 2(a), lane 7). Two different types of circular oligomers were observed that yielded a single 4.36 kb linear fragment on digestion with XhoI (Fig. 2(a), lanes 8 and 10); one yielded 0.78 kb and 3.59 kb fragments after digestion with XhoI +PstI (Fig. 2(a), lane 9) and the other yielded 2.02 kb and 2.34 kb fragments after digestion with XhoI+PstI (Fig. 2(a), lane 11). These results are consistent with the two types of circular oligomers containing only either pRDK35 tet-10 or pRDK39 tet-14 as repeating monomeric units, respectively (Fig. 3). A third type of oligomeric species was obtained which yielded 1.25 kb and 3.1 kb fragments on digestion with XboI (Fig. 2(a), lane 12) and yielded 0.78 kb, 1.25 kb and 2.34 kb fragments on digestion with XhoI + PstI (Fig. 2(a), lane 13). These results are consistent with the circular oligomers having a repeating monomeric

Table 3 of Escherichia coli with diflerent

Transformation

(v/o) b

Recombination

AU1 157

JCS679

A131157

oao9 NT’ 0.014 ow9 NT NT

0.79 0.44 0.94 0.91 0.41 0.51

2.8 NT 10.7 2.3 NT NT

linear frequency

dimers (%)b

obtained

with

JC8679 8.1 7.2 13.1 16.0 1.8 36.1

Substrate DNAs and transformation assays were as described under Methods. In each experiment the circular control DNA was the uncleaved circular version of the linear substrate. a Calculated exactly as described in Table 1. b Calculated exactly as described in Table 2. c NT, not tested.

DNA

JC8679 JC8679 AU1 157 AU1157 JC8679 JC8679 AS1157 AU1 157 JC8679 JC8679 JC8679

AP’ TC’ AP’ Tc’ AP’ TC’ AP’ TC’ AP’ AP’ AP’

Selection 14 12 17 7 31 19 12 13 29 29 20

NC

analysis

30.8 20.7 3.4 25.0

89.4

14.3 100 11.8 100

pBR322

of plasmid

65.5 55.0

-. -

50.0 -d 5.9

pRDK35tet-IO

Single

individual

10.0

48.3

5.1)

17.6

31.0 13.8

.~

14.3

pBR322tet-10,

products

tet-14

observed

transformants

21.4

pRDK39tet-14

genotypes

Recombination

from

monomer

puri$ed

Table 4 DNA

’ Plasmid DNA was purified from individual transformants and analyzed as described under Methods and in the legend to Fig. 2. b The structures of the plasmid DNAs observed are as described in Figs 1 and 3 and by Doherty et al. (1983). ’ Number of transformants tested. * ~, not observed. e pUR322 + pUR322tet-10, t&l4 (5.3%) and pBR322 + pRDK35tet-10 (5.3%). f pRDK35tet-IO + pSR322tet-10, w-14 (17.3%). g pSR322 + pRDKtet-10 (10%). h Dimer of pBR322 + pRDK35tet-IO (30.8%), d’lmer of pSR322 + pBR322tet-10, tet-14 (15.4%) and dimer of pBR322 (23%).

pRDK4l/BamHI pRDK41/BamHI pRDK41/BamHI pRDK41/BamHI pRDK41 pRDK41 pRDK41 pRDK41 pRDK68jXbaI pRDKGS/XbaI pRDK’Il/XbaI

Substrate

Strain tested

Structural

17.3’ 10.0 g

10.6 e

Multiple monomer genotypes

(%) b

a

100.0

100.0

58.8

pRDK41

Dimer

69.2 ’

Other

genotypes

520

L. S. Symington,

P. Morrison

BornHI-Xbul pRDK 69

Figure 1. Restriction endonuclease cleavage site maps of recombination substrates. A map of pRDK41 is presented and the positions of the XbaI linkers that were inserted during the construction of pRDK68, pRDK69. pRDK70 and pRDK71 are indicated on the map.

unit of the pBR322 tet-10, tet-14 double mutant (Fig. 3). The Tc’ recombinants obtained when E. coli JC8679 recBrecCsbcA was transformed with circular pR,DK41 all contained a series of circular oligomers that were resistant to digestion with XhoI, and yielded 4.36 kb linears after digestion by PstI (data not shown). Thus, the repeating monomer unit of these oligomers is wild-type pBR322. The plasmid DNAs obtained after transformation of E. coli JC8679 recBrecCsbcA with circular pRDK41 and selection for Ap’ had circular dimers as the major species ( >90°/o; data not shown). Digestion of this DNA with XhoI yielded 3.1 kb and 5.6 kb fragments while digestion with XhoI + Pstl yielded 0.78 kb, 2.02 kb, 2.34 kb and 3.58 kb fragments, indicating that most of the DNA present in these transformants was identical with pRDK41 (Fig. 2(a): lanes 1 to 3 and Fig. 1). These latter results were identical with our previously published results on the recombination of pRDK41 in E. coli JC8679 (Doherty et al., 1983). The structure of the plasmids obtained after transformation of E. coEi AB1157 by linear dimer DNA was also studied. Two types of Ap’ transformants were obtained; 590/b were circular dimers (Fig. 2(b), lane 1) and 41% were circular monomers (Fig. 2(b), lane 5). Digestion of all of the circular dimers with BamHI produced a single 4.36 kb linear fragment (Fig. 2(b), lane 2). All of the circular dimers yielded 3.1 kb and 5.6 kb fragments after digestion with XhoI (Fig. 2(b), lane 3), and they yielded 0.78 kb, 2.02 kb, 2.34 kb and 3.58 kb fragments after digestion with XhoI and PstI (Fig. 2(b), lane 4). These data indicate that all of these dimers had the same structure as pRDK41 (Fig. 1). Four types of circular monomers were obtained. One monomer (Fig. 2(b), lane 5) yielded a 4.36 kb linear species on digestion with XhoI (Fig. 2(b), lane 6) and yielded 0.78 kb and 3.58 kb fragments after digestion with XhoI and PstT (Fig. 2(b). lane 7). Thus, this particular monomer

and R. Kolodner

appears to be identical with pRDK35 tet-10 (Fig. 3). The other monomeric species obtained (data not shown) appeared to be identical with either pBR322, pRDK39 tet-14 or the pBR322 tet-10. tet-24 double mutant (Fig. 3). When the Tc’ transformants were analyzed, all of the plasmids were circular monomers that appeared to be identical t,o wild-type pBR322 (data not shown). Transformation of E. coli AH1157 with circular pRDK41 only yielded circular dimers t’hat appeared to be identical with pRDK41 when Ap’ transformants were selected (data not shown). When Tc’ transformants were selected aft’er transformation of E. coli AB1157 with circular pRDK41. 31% of the transformants contained wild-type pBR322 monomers. The remaining Tc’ transformants contained circular dimers in which one of the tet genes was wild type and the other tet gene was either a wild-type gene, a tet-10 single mutant gene or a tet-10, tet-14 double mutant gene (data not shown). These latter results are identical wit’h our previous results on the recombination of pRDK41 in E. coEi AB1157 (Doherty et al., 1983). The results obtained from a number of different experiments are given in Table 4. These data, along with t’he results presented in Tables 1 and 2, show that the products of transformation of E. coli by linear dimer plasmid DNA differ depending on which recombination system was funct’ional in the recipient strain. In wild-type strains, linear dimers transform very inefficiently, wit’h about’ 400, of the transformants being associated with the AP’ formation of a monomeric recombinant, and about SOY0 of the transformants being associated with the formation of the original circular dimer substrate. The latter molecules could be due eit’her t)o religation of the linear dimer after transformation or by transformation with contaminating uncut plasmid DNA. Transformation of E. coli JC8679 recBrecCsbcA, in which t)he RecE recombination system is active. by linear dimer DNA was ver) efficient and all of resulting Ap’ t’ransformant)s contained recombinant monomer units. In seven transformants (6.77;) obtained after transforming different linear dimers into E. co& JC8679 recBrecCsbcA . variable amounts of two different repeating monomeric units were observed within an individual transformant. Their origin is not clear. but) t,hey could represent double transformants or could have resulted from the replicat,ion of a heteroduplex recombination product prior to mismatch repair. When circular dimers were transformed into either strain and 9p’ was selected. the resulting transformants contained primarily the initial circular dimer. whicsh is considerably different than when linear dimers were transformed into each strain. Transformation of either E. coli AR1 157 OI JC8679 recBrec(lnbcil with linear dimers. followed by selection for Tc’. only yielded wild-type pBR322 DSX. although at a IOO-fold lower yield in wildt)ype E. coli t)han in JC8679 (Table 2). Selection for Tc’ aft’er transformation with circular dimers, as

Intramolecular I

2

3

4

Recombination

5

7

6

521

of Linear DNA 8

9

IO

II

12

I3

(a)

Tetramer Trimer Dimer

5.60 4.36 3 59 3.10

Monomer

2.34 2.02 I.25 0,78

I

2

3

4

5

6

7

(b)

5.60

Dimer

4.36 3.59 3.10 ier

2.34 2.02

0.78

Figure 2. Electrophoretic analysis of plasmid DNA purified from individual transformants. (a) Analysis of transformants with linear dimers ofpRDK41 obtained from a BamHI partial digest. obtained by transforming E. coliJC8679 recBrecCsbcA (‘ircular pRDK41 marker DPU’A undigested (lane l), digested with XhoI (lane 2) and digested with XhoI and PstI (lane 3). Tetracvclinr-resistant plasmid undigested (lane 4), digested with XhoI (lane 5) and digested with XhoI and P&I (lane 6). Ampicillin-resistant plasmid undigested (lane 7), digested with XhoI (lane 8) and digested with XhoI and P&I (lane 9). Two additional ampicillin-resistant plasmids digested with XhoI (lanes 10 and 12, respectively) and digested with XhoI and PatI (lanes 11 and 13, respectively). (b) Analysis of transformants obtained by transforming E. coli AB1157 with linear dimers of pR,I>K41 obtained from a BumHI partial digest. Ampicillin-resistant plasmid undigested (lane l), digested with BamHI (lane 2), digested with XhoI (lane 3) and digested with XhoI and PstI (lane 4). Tetracycline-resistant plasmid undigested (lane 5), digest,ed with XhoI (lane 6) and digested with XhoI and PstI (lane 7). The numbers at the right of the Figure are the sizes. in kb. of one of the indicated DNA fragments. The fragments were identified by comparison wit’h authentic samples prepared by digestion of appropriate plasmids (Doherty et al., 1983).

compared to transformation with linear dimers, vielded a similar set of recombinants in the case of k. coli JC8679 recBrecCsbcA and a very different set of recombinants in the case of E. coli AB1157. A more detailed examination of recombination of circular pRDK41 in these E. coli strains has been published elsewhere (Doherty et d., 1983). The position of the double-strand break in the linear dirner substrates affected the distribution of Ap’ recombinants obtained after transformation

recBrecCsbcA. In the case of of E. coli JC8679 pRDK68 and pRDK69, where the double-strand break is in the tet gene and produces recombination substrates containing both tet mutant gene segments in one half of the substrate, we found that the single mutant recombination product that was present closest containing the tet mutation to the end of the substrate was not formed. The effect of the position of the double-strand break on was not examined recombination in E. coli AB1157

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L. S. Symington, P. Morrison

pBR322

pRDK39

35

kb

Pvull

Pvull pRDK

0.5

pBR322tel-lo,&-/4

Figure 3. Restriction endonucleasecleavagesite maps of monomeric recombinant genomes.All are identical with pBR322, except that they contain the indicated XhoI linker insertion mutations.

in detail because none of the linear dimers tested transformed this strain efficiently (Table 3).

4. Discussion We have studied the transformation of E. coli by linear dimers of pBR322 that contain two different mutant tetracycline resistance genes. Transformation of a wild-type E. coli strain and an isogenic recBrecCsbcA E. coli strain were studied because these strains catalyze plasmid recombination events by a different recombination system. Our results indicate that the establishment of circular replicating plasmids after transformation of wild-type E. coli with linear dimers involves one of three mechanisms: intramolecular recombination to produce a recombinant circular monomer, religation to produce the original circular dimer or transformation with contaminating circular dimers that might be present at low levels in the substrate DNA. In contrast, transformation of E. coli JC8679 recBrecCsbcA appeared to be mediated exclusively by intramolecular recombination of the linear dimer substrate DNA to form recombinant monomers. Conley & Saunders (1984) have also studied the transformation of wild-type E. coli by linear dimers observed transformation of pBR322 and frequencies that were 25% of those obtained with circular dimers but did not provide any structural information about the plasmids present in their transformants. Their results were different from ours for reasons that we do not yet understand. Transformation of wild-type E. coli strains with linear dimer DNA was inefficient (< 1%) compared to transformation by circular dimer DNA, which is consistent with the observation that plasmid recombination in wild-type E. coli strains only

and R. Kolodner

occurs at frequencies of 10e3 to 10e4 per generation (Fishel et al., 1981; James et al., 1982: Laban & Cohen, 1981). The effect of different ret mutations on the generation of Tc’ recombinants after transformation with linear dimer DNA was similar to the observed effects of these mutations on recombination of circular plasmids. These results suggest that recombination of linear and circular substrates in wild-type E. coli strains occurs by a similar mechanism. The observation that none of the ret mutations decreased the frequency of transformation by linear dimers is not surprising. because approximately 600% of the transformants appear to arise by a mechanism that is unrelated to recombination. Recent studies with yeast have demonstrated that the presence of double-strand breaks in DNA can be highly recombinogenic (OrrWeaver et al., 1981; Szostak et al., 1983). In our experiments, the frequency of obtaining Tc’ recombinants was increased 50 to loo-fold when circular dimers were linearized prior to transformation into wild-type E. coli. However, it is not clear if this is due to a stimulation of the RecF pathway of recombination by double-strand breaks. The actual yield of Tc’ recombinants was slightly lower with the linear substrates as compared to the circular substrates. This can be explained if the frequency of recombination of both linear and circular dimers is similar and that only those linear dimers cyclized by recombination are established as replicating plasmids after transformation, whereas, all of the circular dimers can give rise to viable transformants. The preferential selection against the establishment of non-recombinant linear plasmids is most likely a result of exonucleolytic degradation of the linear DNA, compared to circular DNA, following transformation. Transformation of recBrecCsbcA E. coli strains with linear dimer DNA is very striking in that linear dimers transformed these strains with the same efficiency as circular dimers and almost all of the resulting transformants contained a series of circular oligomers having a single repeating recombinant monomeric unit. This observation suggests that, when linear dimers enter the recipient, they cyclize by intramolecular recombination to form a recombinant monomer that subsequently participates in additional recombination events to form circular oligomers during the growth of the transformant. The high frequency of recombination observed is consistent with the observation that sbcA mutations induce an efficient system for plasmid recombination and that linear DNA is the preferred substrate for exonuclease VIII, an enzyme that appears to be required for the sbcA-induced (RecE) system (Fishel et al., 1981; Laban & Cohen, 1981; James et al.. 1982; Joseph, 1983; Joseph & Kolodner, 1983a,b). Genetic analysis indicates that recA and recF mutations have no effect on transformation by linear dimers, whereas recE mutations reduce both the frequency of transformation and the generation of Tc’ recombinants. This is consistent with the

Intramolecular

Recombination

observed effects of these mutations on the recombination of circular plasmids in recBrecCsbcA E. coli strains and suggests that both recombination reactions occur by a related mechanism (Cohen & Laban, 1983; Fishel et al., 1981; James et al., 1982; Joseph, 1983; Laban & Cohen, 1981). The failure of the recE159 mutation to have a larger effect than that observed could be explained if it was a leaky mutation, or if another nuclease such as exonuclease III can partially satisfy the requirement for exonuclease VIII. Whether the recombination of circular plasmids initiates with the production of a double-strand break is not addressed in this study and will be the subject of further investigation. The intramolecular recombination of linear dimers by the RecE recombination system can be explained by several different mechanisms, including two t’hat involve the action of exonuclease VIII at the ends of the linear substrate. Any viable mechanism must involve the formation of regions of heteroduplex DNA and subsequent gene conversion in order to explain the formation of Tc’ recombinants from linear pRDK70 DNA. Three such mechanisms are illustrated in Figure 4. In the first mechanism, one end of the molecule is partially degraded by exonuclease VIII to produce an end having a, 3’ single-stranded overhang. This singlestranded end subsequently pairs with a homologous region in the middle of the molecule and then forms

5’

Model

I

5’

of Linear DNA

523

a D-loop similar to that proposed in a number of pairing schemes (Meselson & Radding, 1975; Radding, 1981). The resulting heteroduplex region could then be extended by branch migration. At any point, the molecule could be processed by the action of appropriate nucleases to form a partially heteroduplex monomer or branch migration could proceed further until a fully heteroduplex monomer was formed. In an alternative processing scheme, the displaced strand of the D-loop is nicked and allows a Holliday junction to be formed, which must then be resolved to yield a heteroduplex monomer (Holliday, 1964). Any mismatched nucleotides present could be processed by a mismatch repair reaction or by DNA replication (Doherty et al., 1983). If both ends are degraded and both ends initiate recombination by strand invasion at the middle of the linear dimer, then recombination could resemble the double-strand break repair model of recombination (Szostak et al., 1983). This mechanism could also explain the rare production of multiple monomer genotypes in a single transformant that we observed (Table 4). In the second model, two homologous duplex regions pair and initiation leads to the formation of a Holliday junction (Holliday, 1964). Branch migration and resolution lead to the formation of a partially heteroduplex monomer that can then be repaired as discussed above. The first t’wo models

Model

2

5’

Model

3

T-iI (b)

5’-G-

I Cd)

\ (e)

I

(h)

J

(e)

Figure 4. Models for the recombination of linear dimers. Thick and thin lines are used to designate the two different monomer units of the linear dimer. Potential steps in the models are: (a) exonuclease VIII digestion; (b) homologous pairing: (c) annealing of homologous single strands; (d) branch migration; (e) digestion with single-stranded DNAspecific endonuclease; (f) digestion with single-stranded DKA-specific nuclease; (g) strand exchange similar to that proposed in the original Holliday model (Holliday, 1964); (h) resolution of the Holliday junction; and (i) isomerization, which is the rotation of complementary strands around each other. Note: in model 3, asymmetric digestion of one strand past the middle of the molecule combined with annealing and some repair synthesis will yield heteroduplex monomers that are identical with those formed in models 1 and 2. Exonuclease VIII digestion in viva is potentially asymmetric due to its processive mode of action (Joseph & Kolodner. 19836).

524

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P. Morrison

are very similar, with respect to the products they yield; however, the second model does not have an obvious requirement for exonuclease VIII. In the third model, extensive degradation by exonuclease VIII at both ends occurs to produce a molecule having two 3’ single-stranded ends. Once degradation exposes a region of homology on both sides of the molecule it reanneals to form a partially heteroduplex circular monomer that has two singlestranded tails. The heteroduplex region can be extended by additional degradation or by branch migration with the resulting single-stranded tails being removed by digestion with a single-stranded DNA-specific nuclease. Mismatched nucleotides are then repaired as discussed above. A model that is similar to the third model has been used to explain the recombination of linear plasmid DNAs containing tandem duplications after introduction into mammalian cells (Lin et al., 1984). It is difficult to distinguish between these models on the basis of our results, because by altering the factors that affect heteroduplex formation (extent of degradation, initiation site, branch migration direction) each model can be used to predict the formation of the observed products. Furthermore. each model predicts that’ the products that were not observed after recombination of linear pRDK68 and pRDK69 and the formation of wild-type monomers by recombination of linear pRDK70 will be quite rare. This is because the only way to produce these species is by the formation of a gene that is heteroduplex at both mutant sites followed by appropriate independent’ mismatch repair events. Given that the frequency of independent) repair events is 1 to 2%, our sample size (Table 4) was not large enough to observe these species by the analysis of individual transformants (Fishel & Kolodner, 1983). We find the third model attractive since it utilizes exonuclease VIII and would probably not require a homologous pairing protein because intramolecular reannealing of homologous single strands should be very favorable. This is consistent with the observation t’hat the RecE recombination system is recA-independent (Fishel et al., 1981; Laban & Cohen, 1981; this study). sbcA mutations could induce t,he Alternatively, synthesis of a recA protein analog. Clearly, further enzymological and genetic studies will be required to determine the exact mechanism of the RecK recombination syst’em. The authors thank Drs Richard A. Fishel and Cynthia A. Luisi-DeLuca for their comments on the manuscript. and Maryellen Thomas for her help in preparing the manuscript. This work was supported by NH grants GM29383 and GM26017, and ACS grant FRA-271 to R.K. L.S.S. was a postdoctoral fellow of the Damon Runyon-Walter Winchell Cancer Fund.

C’arter. D. M. & Radding, (1. M.

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194, 21 l-218. Doherty, M. J., Morrison, P. T. & Kolodner. R. 1). (1983). J. %ol. Niol. 167, 539-560. Emmerson, P. T. & Howard-Flanders, I-‘. (1967). *J. Racteriol. 93, 172991731. Fishel, R. A. & Kolodner, R. (1983). In UCLA Symposia on Molecular and Cellular Biology (Friedberg. E. C. & Bridges. B. A., eds); vol. 1 I. pp. 309-324. Alan R. Liss, Inc.. New York. Fishel, R. A. & Kolodner, R. (1984). J. Bacterial. 160, 116881170. Fishel, R. A., ,James, A. A. & Kolodner, R. (1981). AVature (London), 294, 184-186. Gillen. .J. R. & Clark, 4. J. (1974). In Mechanisms in tlecombination (Grell, R. F., ed.); pp. 123-135. Plenum Publishing Corp., Kew York. Gillen, .J. R.. Karu, A. E., Nagaishi, H. & Clark. A. .J. (1977). J. Mol. Biol. 113. 27-41. (Zllen. .J. R.. iVillis, D. K. & Clark, A. -J. (1981). J. Hacteriol. 145, 521b.532. Holliday. R. (1964). Genet. Rea. Camb. 5, 282-304. Holmes. 1). S. & Quigley, M. (1981). Anal. B&hem. 141. 193196. Horii. %. 1. & (‘lark. A.
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