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

Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes (exonuclease/germinal vesicle/nudeoside triphosphates/single strand aneling)

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

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

Xenopus laevis oocytes efficiently recombine ABSTRACT linear DNA injected into their nuclei (germinal vesicles). This process.requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5' -+ 3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms.

nique and have led to the widespread use of oocyte and egg extracts. Oocyte extracts have been shown to support transcription (22, 23), chromatin assembly (24, 25), and DNA repair (26), whereas egg extracts perform DNA replication (27, 28), mismatch repair (29), DNA end joining (30), and modification of RNA duplexes (31). Extracts made from Xenopus eggs and oocytes also appear to catalyze the formation of branched (32, 33) and catenated DNA structures (34, 35), which may be recombination intermediates. Our efforts to establish oocyte extracts capable of performing homologous recombination were encouraged by the above studies. We show here that homogenates of manually isolated oocyte nuclei support complete recombination events in vitro, and we report some requirements of the process.

MATERIALS AND METHODS Germinal Vesicle (GV) Extract Preparation. X. laevis ovary segments were surgically removed (36); oocytes were isolated by collagenase digestion (16), rinsed extensively in OR-2 medium (37), and kept at 19'C in OR-2 medium until dissected (a few hours to 2 days). Healthy stage VI oocytes (38) were selected and rinsed immediately before use in supplemented isolation buffer (39), which is composed of 20 mM Tris HCI (pH 7.5), 75 mM KCI, 7 mM MgCl2, 2 mM dithiothreitol, 2% polyvinylpyrrolidone (PVP-360; Sigma), and 0.1 mM EDTA. For studies of divalent cation requirements, isolation buffer was made without MgCI2. Each GV was manually removed by using watchmaker's forceps in isolation buffer at room temperature and transferred in 0.5-1 A.l to a tube kept on ice. After several hundred GVs had been collected in a few hours, they were disrupted by pipetting repeatedly through a 200-pl micropipet tip. Particulates were sedimented at 500 x g for 5 min at 40C, and aliquots of the supernatant were frozen in liquid N2 and stored at -70'C. Activity was retained for at least 6 months. DNA Substrates. pRW4 (18) consists of pBR322 (40) with a tandem duplication of the sequences from position 25 to position 1270, separated by a unique Xho I site. Plasmid DNA was isolated by banding in ethidium bromide/CsCl density gradients (18). Preparative restriction enzyme digests with Xho I were performed as recommended by the manufacturers (Boehringer Mannheim or Bethesda Research Laboratories). Digests were stopped by adding EDTA to a concentration of 25 mM, extracted with phenol/chloroform/isoamyl alcohol (25:24:1 vol/vol), and then extracted with water-saturated ether. The DNA was precipitated by adding 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol and then collected by centrifugation. Pellets were dissolved in 10 mM Tris HC1, pH 8.0/1 mM EDTA at 1 ng/lul.

Homologous genetic recombination is crucial for meiosis and also occurs in mitotic cells. Because of its role in chromosome segregation, allele redistribution, and DNA repair, recombination has attracted the attention of a wide variety of investigators (for reviews, see refs. 1-3). A focus for these studies recently has been the molecular mechanisms of recombination reactions. Much of our current knowledge is derived from the analysis of fungal recombination products and the characterization of phage and bacterial recombination enzymes (4-6). Further progress would be facilitated by the development of in vitro systems that can support complete recombination events. A number of organisms have been used for this purpose. Extracts and partially purified components capable of catalyzing at least partial events have been prepared from Escherichia coli (7, 8), Ustilago maydis (9), Saccharomyces cerevisiae (10, 11), and mammalian cells (12-15); however, recombination products were generated only at low levels, which have usually required amplification by bacterial transformation. We have been studying the very efficient homologous recombination of DNA molecules injected into Xenopus laevis oocyte nuclei (16-20). This process is dependent on homology and on molecular ends, and an early step is resection by a 5' -* 3' exonuclease. It appears to proceed by a nonconservative pathway that is analogous to some types of mitotic recombination observed in other organisms (19). Xenopus oocytes are large, easily manipulable cells that have an internal volume and quantities of many cellular components equivalent to 105 somatic cells (21). These characteristics have made oocyte injection a popular techThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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

10840

Biochemistry: Lehman and Carroll

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

10841

Oligo #1

Oligo#2 I

3.54 2.07

..

'4

Y...

II

Y

.

=

Substrate

III

IV 4.14

.. ,, I.

5.61

.7- .. i

7.08

*--

..

_ ,-B..

_

,

=_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 boxes with 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 in analyzing 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 covalently

closed product. The competing side reaction shown in step IV yields three end-joined products. The oligonucleotide probes used in Fig. 3C are complementary to sequences at the left end of the repeats as drawn and are represented above their homologous sequences, with 3' ends shown as half arrowheads.

Recombination Reaction. For a standard 7-,ul reaction, the was thawed on ice and 5 pul, representing 5-10 GVs, was used in each reaction. One microliter of DNA solution (1 ng) and 1 pLI of supplements (salts, nucleotide triphosphates, etc., as appropriate) were added to the extract, and incubation was carried out overnight at room temperature. dNTPs (Boehringer Mannheim) and rNTPs (Pharmacia LKB) were obtained as sodium salts. The nonhydrolyzable analogues adenosine 5'-[y-thio]triphosphate (ATP-[y-S]) and adenylyl imidodiphosphate (AMP-PNP) were obtained as tetralithium salts (Boehringer Mannheim). The reactions were stopped by the addition of 2 .ul of 10% SDS, 1 ,ul of tRNA (10 ,gg/pl), and 90 tLI of 150 mM NaCi/10 mM Tris HCl, pH 7.5/5 mM EDTA. The samples were extracted, precipitated, and redissolved as above. Analysis of DNA. Recovered DNA was digested with the diagnostic restriction enzyme Pvu II (or analyzed uncut as in Fig. 2). The samples were then subjected to electrophoresis in horizontal 1.2% agarose gels. Alkaline gels were run as described by Sambrook et al. (41). The DNA was depurinated with 0.25 M HCl prior to transfer in 0.4 M NaOH to Zeta-Probe nylon membranes (Bio-Rad). Most blots were hybridized with random-primed (42) linear pRW4 having specific activities of 0.5 x 109-3.0 x 109 cpm/pLg of DNA. The blot shown in Fig. 3C was stripped with 0.2 M NaOH and rehybridized with oligonucleotides labeled with T4 polynucleotide kinase (New England Biolabs), having specific activities of 1.0 x 107-1.2 x 107 cpm/pmol. The oligonucleotides correspond to pBR322 sequences 63-78 (no. 1), and extract

78-63 (no. 2) (both 5'

--

3'). [a-32P]dCTP and

Initially we surveyed a variety of conditions in attempts to reconstitute homologous recombination in whole oocyte extracts. Buffers optimized for 5S RNA transcription (22, 44) or chromatin assembly (24) were used in preparing high-speed supernatants from intact oocytes. Although RNA polymerase, topoisomerase, and exonuclease activities were present, none of these preparations generated detectable recombination products (data not shown). Because the activities involved in recombination are localized in the nucleus, we next made extracts from manually isolated nuclei. Fig. 2 shows the result of incubation of the linear 5.61-kb recombination substrate in the presence or absence of GV extract prepared in isolation buffer (39), with added dNTPs and rNTPs. The extract catalyzed the formation of high molecular weight multimeric species and topoisomers of circular pBR322 (uncut, " + Extract" lane in Fig. uncut PvuIl Extract + + -

-

7.08 5.61

_ _

EJ EJ

4.36

w

p EJ

4.14

[y-32P]ATP

3.54

purchased from Amersham. Hybridization and washing of the membranes was as described by Church and Gilbert (43).

_

S

were

RESULTS The substrate used to assay for recombination is illustrated schematically in Fig. 1. It is a linear DNA molecule (pRW4/ XhoI) with terminal direct repeats 1246 base pairs (bp) in length. Intra- or intermolecular recombination can occur within these repeats to produce circular monomers and linear or circular multimers of pBR322 (16, 18). Products were usually analyzed by digestion with Pvu II, which cuts once within the plasmid sequence, outside the homologous overlaps (triangles in Fig. 1). This generates one 4363-bp linear fragment for each inter- or intramolecular recombination event that has occurred.

2.07

S

FIG. 2. Formation of recombinants is dependent on the presence of the GV extract. One nanogram of linear pRW4 was incubated overnight in a 7-1.l mixture containing each dNTP at 1 mM, each rNTP at 0.5 mM, and extract from six GVs (+) or isolation buffer without GV extract (-). Both undigested (uncut) and Pvu II-digested samples are shown. For the digested samples, S indicates the position of substrate fragments, P denotes the products of homologous recombination, and EJ indicates end-joined bands. Linear DNA sizes on the left are given in kb. Hybridization was with a pRW4

probe.

10842

-l~

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

Biochemistry: Lehman and Carroll

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

7 . ()

5..6 I 4.14 .i

54-

()

().I (0.

2

( ( 0

4_M

05 .2

n.\1 d\ l}P" ). 1> Ii 1" ;1.-

() ()z

!' '

.F

.

)

.I

_0 R7

P r b e:

pRW4

p

R

W

Alkaline

4

pR

4

O)ie*

()Iigu

Gel

FIG. 3. Dependence of recombination on added dNTPs. Each sample contained 1 ng of DNA, extract from six GVs, and the indicated final concentrations 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 by electrophoresis. 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 gel electrophoresis of samples from a different experiment than those in A, probed with pRW4. (C) Neutral gel electrophoresis of samples from a third experiment. Sequential hybridizations were performed with pRW4 and oligonucleotide probes 1 and 2 (see Fig. 1), as indicated. The arrow at the right indicates the smear of degraded substrate fragments that is diagnostic for exonuclease polarity. It hybridized with oligonucleotide no. 1 but not oligonucleotide no. 2.

2). The hybridization signal of these products appears low in Fig. 2, because the products are distributed among many separate species and also due to less efficient transfer of high molecular weight products. Upon Pvu II digestion this material largely ran as 4.36-kb product (designated P). In addition, a small amount of 3.54and 2.07-kb substrate fragments (designated S) remained after overnight incubation. There was also a low level of end-joined products (designated EJ,

see

below) produced.

The ratio of high molecular weight products to monomer circular products in the uncut sample reflects the balance between inter- and intramolecular reactions. This ratio varied somewhat among experiments, as is observed in vivo (45). Incubation in isolation buffer alone did not alter the substrate DNA ("- Extract" lanes in Fig. 2). Our first recombination reactions were incubated in the presence of both rNTPs and dNTPs because we anticipated possible requirements for energy and RNA or DNA synthesis. When we examined these requirements individually, we found that millimolar amounts of dNTPs without rNTPs were able to support efficient recombination (Fig. 3A). In these experiments, recombination of 1 ng of substrate was essentially complete after an overnight incubation with 2 mM dNTPs in extract derived from about six GVs. Analysis of a different experiment by alkaline gel electrophoresis showed that a majority of the products were complete (i.e., consisted of covalently closed strands) (Fig. 3B). Although the samples in Fig. 3 A and B are not directly comparable, it is clear that both

homologous recombination (P)

and

homologous overlaps, leaving gaps. This is different from what is seen in vivo, where the exonuclease does not produce gaps. This difference is interpreted as a block in the resolution of the intermediates into products without added NTPs. As noted in the Discussion, we cannot conclude that formation of these intermediates is NTP independent, since it is possible that they formed during recovery of the DNA and not 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 and oligonucleotide probing, have been used to show that these are the products of joining the substrate ends (see Fig. 1) (unpublished results). End joining only occurred with low levels of dNTPs, but not at any concentration of individual or mixed rNTPs (data not shown). The Xho I site was not regenerated in end-joined molecules,

so

they did

not result

from simple ligation. Sequence analysis of the junctions will be required to determine if the end joining is dependent on short terminal homologies (46). A

ml N1

.; 3f0

ATP-y-S AMP-PNP 10 52) 0Q.1 0 .520

IiT

iAXE

N

k

I

dAIT

52

41

4_

end-joined (EJ)

products were covalently closed. Even in the absence of added dNTPs, the oocyte exonuclease (18) appeared to be active (Fig. 3A, 0 mM). This was evident in the partial degradation of substrate fragments (smears trailing below the 3.54- and 2.07-kb bands). Also the smear beginning above the position of products is characteristic of intermediates with annealed junctions containing unassimilated single-stranded tails (see Fig. 1) (19). The intermediate smear continued below the product band at low levels of dNTPs (Fig. 3, 0 and 0.1 mM), suggesting that the exonucleolytic degradation proceeds beyond the ends of the

)

kt

.*

o. M

_w -_

FIG. 4. Recombination reactions with individual NTPs. Conditions were as in previous figures, except that single NTPs or nonhydrolyzable analogues were included at the concentrations indicated. Incubations were overnight, and the samples were digested with Pvu II for analysis and probed with pRW4.

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

Biochemistry: Lehman and Carroll

vitro. The process appears to follow the same pathway that characterizes the homologous recombination of DNA molecules injected into nuclei of intact oocytes (16, 18-20). These in vivo experiments suggest that the pathway consists of the following steps: degradation by a 5' -+ 3' exonuclease until complementary 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 complete assimilation of the 3' ends, and ligation into covalently closed products (Fig. 1, step III). Like intact oocytes, the extracts have a 5' -* 3' exonuclease activity (18), form intermediates with annealed junctions (20), and generate covalently closed product strands (16, 20). Thus, the extracts promise to allow detailed investigation of the requirements for, and mechanism of, homologous recombination in oocytes. A single oocyte can convert 10 ng (about 109 molecules) of injected substrate DNA into recombination products in several hours (16, 18). In vitro, an extract from five GVs requires roughly an overnight incubation to complete recombination of 1 ng of substrate. The recombination capacity of the extracts is therefore