Proc. Natl. Acad. Sci. USAVol. 88, pp. 10840-10844, December 1991Biochemistry

Homologous recombination catalyzed by a nuclear extract fromXenopus oocytes

(exonuclease/germinal vesicle/nudeoside triphosphates/single strand aneling)

CHRIS W. LEHMAN* AND DANA CARROLLDepartment of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132

Communicated by John R. Roth, September 10, 1991 (receivedfor review January 8, 1991)

ABSTRACT Xenopus laevis oocytes efficiently recombinelinear DNA injected into their nuclei (germinal vesicles). Thisprocess.requires homologous sequences at or near the molec-ular ends. Here we report that a cell-free extract made fromgerminal vesicles is capable of accomplishing the completerecombination reaction in vitro. Like the in vivo process, theextract converts the overlapping ends of linear substrate mol-ecules into covalently closed products. Establishment of thiscell-free system has allowed examination of the cofactorsrequired for recombination. The first step involves a 5' -+ 3'exonuclease activity that requires a divalent cation but notNTPs. Completion of recombination requires a hydrolyzableNTP; maximal product formation occurs in the presence ofmillimolar levels of ATP or dATP. At submillimolar levels ofall four dNTPs, homologous recombination is inefficient, anda side reaction produces end-joined products. This cell-freesystem should facilitate a step-by-step understanding of anhomologous recombination pathway that operates not only inXenopus laevis oocytes but also in cells from a wide variety oforganisms.

Homologous genetic recombination is crucial for meiosis andalso occurs in mitotic cells. Because of its role in chromo-some segregation, allele redistribution, and DNA repair,recombination has attracted the attention of a wide variety ofinvestigators (for reviews, see refs. 1-3). A focus for thesestudies recently has been the molecular mechanisms ofrecombination reactions. Much of our current knowledge isderived from the analysis of fungal recombination productsand the characterization of phage and bacterial recombina-tion enzymes (4-6). Further progress would be facilitated bythe development of in vitro systems that can support com-plete recombination events. A number of organisms havebeen used for this purpose. Extracts and partially purifiedcomponents capable of catalyzing at least partial events havebeen prepared from Escherichia coli (7, 8), Ustilago maydis(9), Saccharomyces cerevisiae (10, 11), and mammalian cells(12-15); however, recombination products were generatedonly at low levels, which have usually required amplificationby bacterial transformation.We have been studying the very efficient homologous

recombination of DNA molecules injected into Xenopuslaevis oocyte nuclei (16-20). This process is dependent onhomology and on molecular ends, and an early step isresection by a 5' -* 3' exonuclease. It appears to proceed bya nonconservative pathway that is analogous to some typesof mitotic recombination observed in other organisms (19).Xenopus oocytes are large, easily manipulable cells that

have an internal volume and quantities of many cellularcomponents equivalent to 105 somatic cells (21). Thesecharacteristics have made oocyte injection a popular tech-

nique and have led to the widespread use of oocyte and eggextracts. Oocyte extracts have been shown to support tran-scription (22, 23), chromatin assembly (24, 25), and DNArepair (26), whereas egg extracts perform DNA replication(27, 28), mismatch repair (29), DNA end joining (30), andmodification of RNA duplexes (31). Extracts made fromXenopus eggs and oocytes also appear to catalyze the for-mation of branched (32, 33) and catenated DNA structures(34, 35), which may be recombination intermediates.Our efforts to establish oocyte extracts capable of per-

forming homologous recombination were encouraged by theabove studies. We show here that homogenates of manuallyisolated oocyte nuclei support complete recombinationevents in vitro, and we report some requirements of theprocess.

MATERIALS AND METHODSGerminal Vesicle (GV) Extract Preparation. X. laevis ovary

segments were surgically removed (36); oocytes were iso-lated by collagenase digestion (16), rinsed extensively inOR-2 medium (37), and kept at 19'C in OR-2 medium untildissected (a few hours to 2 days). Healthy stage VI oocytes(38) were selected and rinsed immediately before use insupplemented isolation buffer (39), which is composed of 20mM Tris HCI (pH 7.5), 75 mM KCI, 7 mM MgCl2, 2 mMdithiothreitol, 2% polyvinylpyrrolidone (PVP-360; Sigma),and 0.1 mM EDTA. For studies of divalent cation require-ments, isolation buffer was made without MgCI2. Each GVwas manually removed by using watchmaker's forceps inisolation buffer at room temperature and transferred in 0.5-1A.l to a tube kept on ice. After several hundred GVs had beencollected in a few hours, they were disrupted by pipettingrepeatedly through a 200-pl micropipet tip. Particulates weresedimented at 500 x g for 5 min at 40C, and aliquots of thesupernatant were frozen in liquid N2 and stored at -70'C.Activity was retained for at least 6 months.DNA Substrates. pRW4 (18) consists ofpBR322 (40) with a

tandem duplication of the sequences from position 25 toposition 1270, separated by a uniqueXho I site. Plasmid DNAwas isolated by banding in ethidium bromide/CsCl densitygradients (18). Preparative restriction enzyme digests withXho I were performed as recommended by the manufacturers(Boehringer Mannheim or Bethesda Research Laboratories).Digests were stopped by adding EDTA to a concentration of25 mM, extracted with phenol/chloroform/isoamyl alcohol(25:24:1 vol/vol), and then extracted with water-saturatedether. The DNA was precipitated by adding 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of ethanol andthen collected by centrifugation. Pellets were dissolved in 10mM Tris HC1, pH 8.0/1 mM EDTA at 1 ng/lul.

Abbreviation: GV, germinal vesicle; ATP-[y-S], adenosine 5'-[Y-thio]triphosphate; AMP-PNP, adenylyl imidodiphosphate.*To whom reprint requests should be addressed.

10840

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 88 (1991) 10841

I

Y...

Substrate

-& Oligo #1Oligo#2

'4..

II Y . =

III

4.14 ..

5.61 .7-

7.08 *--

IV

,, I. _,-B..

.. i _ , =_g ...

..,

436 -'F- -

Product

End Joined Products

FIG. 1. Model for oocyte recombination. The terminal direct repeats of the recombination substrate (pRW4/XhoI) are indicated by boxeswith arrows showing their orientation. The remainder of the molecules is not shown but is represented by the dashed lines (not drawn to scale).Both intra- and intermolecular recombination events occur, and both are represented by the diagrams. Sites for Pvu II, the enzyme used inanalyzing recombination products, are shown as inverted triangles. Sizes of fragments after Pvu II digestion are indicated [in kilobases (kb)]next to the substrate and products. Step I is catalyzed by a 5' -. 3' exonuclease (generating 3' tails, shown with half arrowheads); step

represents the annealing of exposed, complementary, single-stranded 3' tails; and step III shows the final resolution and ligation into covalentlyclosed product. The competing side reaction shown in step IV yields three end-joined products. The oligonucleotide probes used in Fig. 3C arecomplementary to sequences at the left end of the repeats as drawn and are represented above their homologous sequences, with 3' ends shownas half arrowheads.

Recombination Reaction. For a standard 7-,ul reaction, theextract was thawed on ice and 5 pul, representing 5-10 GVs,was used in each reaction. One microliter ofDNA solution (1ng) and 1 pLI of supplements (salts, nucleotide triphosphates,etc., as appropriate) were added to the extract, and incuba-tion was carried out overnight at room temperature. dNTPs(Boehringer Mannheim) and rNTPs (Pharmacia LKB) wereobtained as sodium salts. The nonhydrolyzable analoguesadenosine 5'-[y-thio]triphosphate (ATP-[y-S]) and adenylylimidodiphosphate (AMP-PNP) were obtained as tetralithiumsalts (Boehringer Mannheim). The reactions were stopped bythe addition of 2 .ul of 10% SDS, 1 ,ul oftRNA (10 ,gg/pl), and90 tLI of 150mM NaCi/10mM Tris HCl, pH 7.5/5 mM EDTA.The samples were extracted, precipitated, and redissolved asabove.

Analysis of DNA. Recovered DNA was digested with thediagnostic restriction enzyme Pvu II (or analyzed uncut as inFig. 2). The samples were then subjected to electrophoresisin horizontal 1.2% agarose gels. Alkaline gels were run asdescribed by Sambrook et al. (41). The DNA was depurinatedwith 0.25 M HCl prior to transfer in 0.4 M NaOH toZeta-Probe nylon membranes (Bio-Rad). Most blots werehybridized with random-primed (42) linear pRW4 havingspecific activities of 0.5 x 109-3.0 x 109 cpm/pLg of DNA.The blot shown in Fig. 3C was stripped with 0.2M NaOH andrehybridized with oligonucleotides labeled with T4 polynu-cleotide kinase (New England Biolabs), having specific ac-tivities of 1.0 x 107-1.2 x 107 cpm/pmol. The oligonucleo-tides correspond to pBR322 sequences 63-78 (no. 1), and78-63 (no. 2) (both 5' -- 3'). [a-32P]dCTP and [y-32P]ATPwere purchased from Amersham. Hybridization and washingof the membranes was as described by Church and Gilbert(43).

RESULTS

The substrate used to assay for recombination is illustratedschematically in Fig. 1. It is a linear DNA molecule (pRW4/XhoI) with terminal direct repeats 1246 base pairs (bp) inlength. Intra- or intermolecular recombination can occurwithin these repeats to produce circular monomers and linearor circular multimers of pBR322 (16, 18). Products wereusually analyzed by digestion with Pvu II, which cuts oncewithin the plasmid sequence, outside the homologous over-laps (triangles in Fig. 1). This generates one 4363-bp linearfragment for each inter- or intramolecular recombinationevent that has occurred.

Initially we surveyed a variety of conditions in attempts toreconstitute homologous recombination in whole oocyte ex-tracts. Buffers optimized for 5S RNA transcription (22, 44) orchromatin assembly (24) were used in preparing high-speedsupernatants from intact oocytes. Although RNA polymer-ase, topoisomerase, and exonuclease activities were present,none of these preparations generated detectable recombina-tion products (data not shown).Because the activities involved in recombination are lo-

calized in the nucleus, we next made extracts from manuallyisolated nuclei. Fig. 2 shows the result of incubation of thelinear 5.61-kb recombination substrate in the presence orabsence of GV extract prepared in isolation buffer (39), withadded dNTPs and rNTPs. The extract catalyzed the forma-tion of high molecular weight multimeric species and topoi-somers of circular pBR322 (uncut, "+ Extract" lane in Fig.

uncut PvuIlExtract - + - +

7.08 _

5.61 _

4.36 w4.143.54 _

2.07

EJ

EJ

pEJS

S

FIG. 2. Formation of recombinants is dependent on the presenceof the GV extract. One nanogram of linear pRW4 was incubatedovernight in a 7-1.l mixture containing each dNTP at 1 mM, eachrNTP at 0.5 mM, and extract from six GVs (+) or isolation bufferwithout GV extract (-). Both undigested (uncut) and Pvu II-digestedsamples are shown. For the digested samples, S indicates theposition of substrate fragments, P denotes the products of homolo-gous recombination, and EJ indicates end-joined bands. LinearDNAsizes on the left are given in kb. Hybridization was with a pRW4probe.

3.542.07

Biochemistry: Lehman and Carroll

10842 Biochemistry: Lehman and Carroll

A Mn diNd,1is B III MdnNT-'P- s

- () ().I (0. 2 ((0 05 .2

7 . ()

5..6 I -l~4.14

.i 54-

R7_0

P r be: pRW4 p R W 4Alkaline Gel

pR 4 ()Iigu O)ie*

FIG. 3. Dependence of recombination on added dNTPs. Each sample contained 1 ng of DNA, extract from six GVs, and the indicated finalconcentrations of each dNTP. The total concentration of dNTPs is thus 4 times that level. The left lane in A and B shows a control sample (-)that was incubated without GV extract or dNTPs. After an overnight incubation, samples were digested with Pvu II and analyzed byelectrophoresis. Bands corresponding to substrate DNA (S), homologous recombination products (P), and the three end-joined products (EJ)are denoted as in Fig. 2. (A) Samples separated by neutral gel electrophoresis and hybridized with the pRW4 probe. (B) Alkaline gelelectrophoresis of samples from a different experiment than those in A, probed with pRW4. (C) Neutral gel electrophoresis of samples froma third experiment. Sequential hybridizations were performed with pRW4 and oligonucleotide probes 1 and 2 (see Fig. 1), as indicated. The arrowat the right indicates the smear of degraded substrate fragments that is diagnostic for exonuclease polarity. It hybridized with oligonucleotideno. 1 but not oligonucleotide no. 2.

2). The hybridization signal of these products appears low inFig. 2, because the products are distributed among manyseparate species and also due to less efficient transfer of highmolecular weight products.Upon Pvu II digestion this material largely ran as 4.36-kb

product (designated P). In addition, a small amount of 3.54-and 2.07-kb substrate fragments (designated S) remainedafter overnight incubation. There was also a low level ofend-joined products (designated EJ, see below) produced.

The ratio of high molecular weight products to monomercircular products in the uncut sample reflects the balancebetween inter- and intramolecular reactions. This ratio variedsomewhat among experiments, as is observed in vivo (45).Incubation in isolation buffer alone did not alter the substrateDNA ("- Extract" lanes in Fig. 2).Our first recombination reactions were incubated in the

presence of both rNTPs and dNTPs because we anticipatedpossible requirements for energy and RNA or DNA synthe-sis. When we examined these requirements individually, wefound that millimolar amounts ofdNTPs without rNTPs wereable to support efficient recombination (Fig. 3A). In theseexperiments, recombination of 1 ng of substrate was essen-tially complete after an overnight incubation with 2 mMdNTPs in extract derived from about six GVs. Analysis of adifferent experiment by alkaline gel electrophoresis showedthat a majority of the products were complete (i.e., consistedof covalently closed strands) (Fig. 3B). Although the samplesin Fig. 3 A and B are not directly comparable, it is clear thatboth homologous recombination (P) and end-joined (EJ)

products were covalently closed.Even in the absence of added dNTPs, the oocyte exonu-

clease (18) appeared to be active (Fig. 3A, 0 mM). This wasevident in the partial degradation of substrate fragments(smears trailing below the 3.54- and 2.07-kb bands). Also thesmear beginning above the position of products is character-istic of intermediates with annealed junctions containingunassimilated single-stranded tails (see Fig. 1) (19). Theintermediate smear continued below the product band at lowlevels of dNTPs (Fig. 3, 0 and 0.1 mM), suggesting that theexonucleolytic degradation proceeds beyond the ends of the

homologous overlaps, leaving gaps. This is different fromwhat is seen in vivo, where the exonuclease does not producegaps. This difference is interpreted as a block in the resolu-tion of the intermediates into products without added NTPs.As noted in the Discussion, we cannot conclude that forma-tion of these intermediates is NTP independent, since it ispossible that they formed during recovery of the DNA andnot during incubation in the extract.At intermediate concentrations of dNTPs (Fig. 3, 0.1 and

0.5 mM), three new products were observed (designated EJ).Several methods, including restriction enzyme mapping andoligonucleotide probing, have been used to show that theseare the products of joining the substrate ends (see Fig. 1)(unpublished results). End joining only occurred with lowlevels ofdNTPs, but not at any concentration of individual ormixed rNTPs (data not shown). The Xho I site was notregenerated in end-joined molecules, so they did not result

from simple ligation. Sequence analysis of the junctions willbe required to determine if the end joining is dependent onshort terminal homologies (46).

A IiT ATP-y-S AMP-PNP dAITml N1 NiAXE 10 52) 0Q.1 0 .520 52

.; 3f0 k 41

I 4_

) kt .* o. M _w -_

FIG. 4. Recombination reactions with individual NTPs. Condi-tions were as in previous figures, except that single NTPs ornonhydrolyzable analogues were included at the concentrationsindicated. Incubations were overnight, and the samples were di-gested with Pvu II for analysis and probed with pRW4.

n.\1 d\ P"l}

() ()z ). 1> Ii 1" ;1.- !' '

4_M . .F

)

.I

Proc. Natl. Acad Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 10843

To determine the polarity of the exonuclease activity seenin Fig. 3A, strand-specific oligonucleotide probes were used.The oligonucleotides are complementary to each other andcorrespond to sequences very near one end of the terminalhomologies that support recombination (see Fig. 1). Theyhybridize far from the end of the 3.54-kb substrate fragmentthat was exposed in the extract, but within 50 bp of theexposed end of the 2.07-kb fragment. In this 2.07-kb frag-ment, oligonucleotide no. 1 is complementary to the strandending 3', and oligonucleotide no. 2 is complementary to thestrand ending 5'. Fig. 3C shows that oligonucleotide no. 1hybridized to both the intact and partially degraded (arrow inFig. 3C) 2.07-kb substrate material. The smear was mostprominent with no added triphosphates. Oligo no. 2 hybrid-ized to the intact 2.07-kb substrate fragment, but not to thesmear of degradation products. These results indicate thatexonuclease activity in the GV extract removed sequences onthe strand ending 5' and, thus, that the exonuclease proceeds5' -- 3', as is seen in vivo (18).

Individual NTPs were examined for their ability to supportthe recombination reaction. As shown in Fig. 4, ATP anddATP gave high yields of homologous products but noend-joined products. The other single rNTPs and dNTPsresulted in substantial, but reduced, product formation (datanot shown). Neither of the nonhydrolyzable ATP analogues,ATP-[y-S] or AMP-PNP, was capable of supporting anydetectable recombination (Fig. 4). This suggests that theNTPs provide an energy source required for the reaction.There was some degradation of the substrate even at highconcentrations of the ATP analogues, indicating that theexonuclease is largely NTP independent.An examination of the requirements for divalent cations in

the recombination reaction is shown in Fig. 5. No recombi-nation was seen in the absence of added divalent cations.Relatively efficient recombination was observed with 5-10mM MgCl2 or 5 mM MnCl2, but higher levels appeared toinhibit the reaction. Neither CaCl2 nor ZnCl2 could substitutefor MgCl2. In fact, the addition of these salts to reactionscontaining MgCl2 completely blocked recombination. NoDNA degradation was apparent in reactions having insuffi-cient divalent cations to support recombination. This sug-gests that the oocyte 5' -* 3' exonuclease activity requiresMg2+ or Mn2' as a cofactor.

DISCUSSIONWe have demonstrated that extracts prepared from Xenopusoocyte nuclei catalyze complete recombination events in

Added CationmM AdditionmM M9CI2 (total)

5.61

_ none Mn- - - - 5 10 25- 5 10 25 - - -

vitro. The process appears to follow the same pathway thatcharacterizes the homologous recombination of DNA mole-cules injected into nuclei of intact oocytes (16, 18-20). Thesein vivo experiments suggest that the pathway consists of thefollowing steps: degradation by a 5' -+ 3' exonuclease untilcomplementary sequences are exposed as single strands (Fig.1, step I), annealing of those strands to form junctions (Fig.1, step II), continued nuclease activity to allow completeassimilation of the 3' ends, and ligation into covalently closedproducts (Fig. 1, step III). Like intact oocytes, the extractshave a 5' -* 3' exonuclease activity (18), form intermediateswith annealed junctions (20), and generate covalently closedproduct strands (16, 20). Thus, the extracts promise to allowdetailed investigation of the requirements for, and mecha-nism of, homologous recombination in oocytes.A single oocyte can convert 10 ng (about 109 molecules) of

injected substrate DNA into recombination products in sev-eral hours (16, 18). In vitro, an extract from five GVs requiresroughly an overnight incubation to complete recombinationof 1 ng of substrate. The recombination capacity of theextracts is therefore <2% of that observed by injecting theDNA into oocytes. We imagine this is due in part to the 10-to 20-fold dilution of nuclear components inherent in themanual isolation of GVs in an aqueous medium. In addition,there may be some leakage during isolation. It is also possiblethat cytoplasmic components are recruited to the nucleusafter injection of large amounts of exogenous DNA in intactoocytes.

Efficient recombination in the extracts requires Mg2+ (orMn2+) and a hydrolyzable NTP. The divalent cation is neededfor exonuclease degradation and possibly for other activities.Optimum yields of homologous recombination products aregenerated with millimolar amounts of ATP, dATP, mixedrNTPs, or mixed dNTPs. These cofactors appear to satisfy anenergy requirement, since nonhydrolyzable ATP analoguesdo not support the reaction.Oocytes have endogenous nucleotide pools (47, 48). As-

suming uniform distribution throughout the oocyte and noleakage or breakdown during GV dissection, we estimate thatthese pools may contribute 10-100 ,M concentrations of therNTPs and <1 uM concentrations of the dNTPs to theextracts. Even if the dNTPs are all nuclear, their concentra-tions in the extracts would be <10 uM. If they are present,the results shown in Fig. 3 indicate that these quantities ofNTPs are insufficient to support complete recombination invitro; however, they may allow a low level ofDNA synthesis.

Ca- . Zn none Mn.a Zn5 10 25 5 10 - - 5 5 5

- - 5 10 5 5 5

EJ

4.36 _ 4 4

3.54 _ - mom

2.07

NM P

;... s

*- s

FIG. 5. Divalent cation requirements for recombination. MgClrfree GV extracts were supplemented with the indicated cations as dichloridesalts. All reactions contained 2 mM dNTPs and were incubated overnight. Samples were digested with Pvu II for analysis and probed with pRW4.

Biochemistry: Lehman and Carroll

10844 Biochemistry: Lehman and Carroll

Nuclease degradation does not depend on added NTPs, butwe cannot say which of the subsequent step(s) is affected.Although intermediates with annealed junctions were foundin the absence of NTPs, it is not clear that they were formedin the extracts, since the annealing might have occurred withextensively resected substrates during extraction and pre-cipitation of the DNA prior to analysis (49). Fig. 3B showsthat in the presence of NTPs, some of the resected singlestrands must have associated in the extract to form completerecombinant products. It would, of course, be of greatinterest to know whether annealing between complementarystrands is catalyzed by an oocyte activity and whether thatactivity requires NTP hydrolysis.

It is likely that the final step in recombination requires NTPhydrolysis, since an ATP-dependent DNA ligase has beenreported in oocytes (35, 50), and the products we find arecovalently closed. The fact that added ATP alone is sufficientfor recombination to proceed to completion suggests thatthere is no requirement for extensive DNA synthesis at anystage. Oocytes contain nucleoside diphosphate kinase activ-ity (51), and we have seen this activity in the extract (data notshown); thus, the terminal phosphate added to the reactionmay be transferred to endogenous nucleoside diphosphates.Therefore we cannot conclude that the NTPs added to thereaction are necessarily the ones utilized for recombination.With low concentrations of added dNTPs, homologous

recombination is inefficient, and a side reaction generatesend-joined products at a modest level. Since dNTPs supportend joining, but rNTPs do not, DNA synthesis may beinvolved in the process. It will be necessary to characterizejunctions of the end-joined products in order to determine ifthey arise by processes previously characterized in oocytes(46) or in egg extracts (30).Both end joining and homologous recombination are ob-

served with DNAs introduced into mammalian cells. Oocyterecombination is very similar to extrachromosomal homolo-gous recombination in mammalian cells (19, 52-56). Both arenonconservative and probably based on exonuclease expo-sure ofhomologous sequences (54). Similar results have beenobtained with plant cells (57) and in some cases with yeast(58, 59). End joints formed in egg extracts share manydetailed features with similarjoints made in mammalian cells(30, 60). Therefore, the information gained by continuedstudy of these processes in Xenopus should be broadlyapplicable.

It would be of interest to isolate and characterize thecatalysts responsible for individual steps in the recombina-tion process. Manually dissected GVs are tedious to prepare,but bulk GV isolation procedures (61, 62) could provide thestarting material for enzyme purification.

We thank Brenda Bass for helpful suggestions concerning NTPsand other aspects, as well as members of our lab for many usefuldiscussions. Thanks are also due to Ed Maryon, Rende Dawson,Genevieve Pont-Kingdon, and Sandra Lehman for helpful commentson this manuscript. This work was supported by a grant from theNational Science Foundation (DCB-8718227). C.W.L. was sup-ported, in part, by a predoctoral training grant from the NationalInstitutes of Health (U.S. Public Health Service Grant 5-T32-GM-07464).

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