Effect of Insertions, Deletions, and Double-Strand Breaks on Homologous Recombination in Mouse L Cells - Semantic ...
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MOLECULAR AND CELLULAR BIOLOGY, Apr. 1985, p. 684-691 Vol. 5, No. 4
0270-7306/85/040684-08$02.00/0
Copyright C 1985, American Society for Microbiology
Effect of Insertions, Deletions, and Double-Strand Breaks on
Homologous Recombination in Mouse L Cells
DAVID A. BRENNER, ANN C. SMIGOCKI, AND R. DANIEL CAMERINI OTERO*
Molecular Genetics Section, Genetics and Biochemistry Branch, National Institute of Arthritis, Diabetes, and Digestive
and Kidney Diseases, Bethesda, Maryland 20205
Received 6 August 1984/Accepted 8 January 1985
We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells.
A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1
thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the
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calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in
hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene
fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for
intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of
recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of
recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before
cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp
increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion
mutant of .24 bp in the same manner did not increase the frequency. We show how our data are consistent
with models that postulate at least two phases in the recombination process: homologous pairing and
heteroduplex formation.
Recombination of DNA sequences in mammalian cells protein level. We then show by using a novel experimental
occurs in germ cells and somatic cells. The analysis of paradigm that the tk+ recombinants arise as a result of
meiotic and mitotic mammalian genetic recombination has homologous pairing between the mutant molecules and not
been hampered by the complexity of the mammalian ge- as a consequence of ligation or single-strand annealing
nome. In somatic cells, examples of recombination events between the cotransferred mutants. Finally, some specific
include immunoglobin gene rearrangements (28, 33), chro- rules of the recombination process are elucidated.
mosomal translocations in cancer (45), and sister chromatid
exchange (16). Recently, general recombination between MATERIALS AND METHODS
two markers on the same chromosome has been demon-
strated in mitotic Chinese hamster ovarian cell hybrids in Plasmid construction. Plasmid pdel9 is a derivative of
culture (41). In the initial studies on homologous recombi- pBR322 created by deleting from nucleotide 1745 to 2505
nation in mammalian cells, infectious viruses such as simian (using the nomenclature of reference 39). M13 tk and pTK
virus 40 and adenovirus were used, but the interpretation of were constructed by inserting a 2.0-kilobase-pair (kbp) PvuII
the experiments was difficult because of the constraints fragment (8) containing the coding region of tk into the SmaI
placed by viral replication and packaging and the possible site of M13mp9 and PUC8, respectively, oriented such that
contribution of viral proteins. We (1, 2) and others (11, 12, the distance from the BamHI cloning site to the BglII site in
30, 38, 44) have studied the recombination of nonhomolo- the tk gene is 256 base pairs (bp). Plasmid pNP, which
gous DNAs transferred into mammalian cells in culture. In contains the 3' end of tk, was constructed by inserting the
this approach, because it is easy to locate the exact positions 2.1-kbp fragment generated by digesting M13 tk with NruI
of the recombinant joints, it is possible to determine the and BglII and ligating this fragment into the NruI and BamHI
sequence and structure of these joints. We have previously sites of pdel9 (Fig. 1). pNP was linearized with NruI, di-
shown that in L cells, the joining of dissimilar DNAs follows gested with the exonuclease BAL 31, and then religated to
a precise scheme (1). DNA-mediated gene transfer can also create the plasmid pNPA35. pNPA35 has a deletion of ca. 35
be used to study homologous recombination between co- bp from the 5' end of the tk fragment contained in pNP, as
transferred DNA fragments of selectable genes (5, 14, 18, 21, determined by polyacrylamide gel electrophoresis. Plasmid
25, 29, 32, 35, 38). pBS, which contains the 5' end of tk, was constructed by
The technique of altering the structure of donor molecules ligating the 1.2-kbp BamHI-SphI fragment of the tk gene into
to study recombination has been successfully used in bacte- the 3.4-kbp BamHI-SphI fragment of pdel9 (Fig. 1A).
ria and fungi (6, 7, 9, 15, 17, 22, 23, 34, 40). To study pST8 was constructed by inserting a nonphosphorylated
homologous recombination in mammalian cells, we con- 8-bp StuI linker into the NruI site of pTK (Fig. 1B). A
structed a family of plasmids with insertions and deletions in HaeIII digest of XX174 replicative form DNA was shotgun
the coding region of the herpes simplex virus type 1 thymi- cloned into the StuI site of pST8, generating pST126, pST318,
dine kinase gene (tk). We show that the tk+ transformants and pST611 (named according to the size of the insert into
isolated from the cotransfer of pairs of mutant tk genes are the original NruI site in tk). XX174 replicative form digested
the result of recombination and not complementation at the with NruI generated a 2,164-bp fragment, which was in-
serted into the Stul site of pST8 to create pST2172. Plasmid
pST72 was created by inserting the 72-bp fragment resulting
*
Corresponding author. from a HaeIII digest of 4X174 replicative form into the NruI
684VOL. 5, 1985 HOMOLOGOUS RECOMBINATION IN MOUSE L CELLS 685
phosphorylated EcoRI (XbaI) Smart linkers (Worthington
Biochemicals Corp.) into the StuI site of pST8 and subse-
LP pBS(4.6 kbp) ))
quently cutting with XbaI and religating. Therefore, none of
c BK PG ENES the pSTI plasmids have a NruI site. pERV was constructed
by digesting pTK with the restriction endonuclease EcoRV,
NES T diluting, and ligating. pERV has a 104-bp deletion in tk that
encompasses the NruI site (Fig. 1). The sizes of the plasmid
insertions were confirmed by polyacrylamide gel electropho-
pNP(5.0 kbp) resis or nucleic acid sequencing or both (20, 31). The
lK .- I
I
f
concentrations of the plasmid DNA were determined both
CH by spectrophotometry and by densitometry measurements
on negatives of photographs of agarose gels stained with
ethidium bromide. The restriction endonuclease digestions
P-.9 of plasmids used for gene transfer were terminated by the
addition of EDTA and heating to 65°C and checked on
agarose gels.
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pB pBS(4.6 kbp)
CKB K P G
)
ENESNj
DNA-mediated gene transfer. DNA-mediated gene transfer
(43) into the recipient Ltk- aprt- mouse cell line was
HB G E IES T performed by the calcium phosphate precipitation technique
JA I Wliiy .1.
and as described in our protocol (4). A 4-,ug sample of each
P",- %
pSTI(4.7 kbp) tk mutant plasmid plus 20 ,ug of Ltk- high-molecular-weight
D Ap carrier DNA were added to 106 cells on a 100-mm plate.
When the uncut plasmid pBS is cotransferred with the uncut
plasmid pST8, the number of tk+ colonies is 63 + 25 (mean
Ap + standard deviation). The cloning of the cell lines and the
C preparation of high-molecular-weight mouse L-cell DNA
were as described previously (4).
( pBS(4.6 kbp) Southern blotting. After digestion with a fourfold excess of
N
C B K P G ENES an appropriate restriction endonuclease(s) (New England
H _ G / BioLabs), 10 ,ug of cellular DNA or 100 pg of plasmid DNA
HB E=
G S was electrophoresed on 1% agarose and transferred by
\1 capillary blotting (36) to a GeneScreen Plus hybridization
Y pER v(4.6 kbp)
transfer membrane (New England Nuclear Corp.). The
Ap membranes were hybridized as described in the protocol of
the manufacturer in formamide at 42°C with nick-translated
[32P]dATP-labeled probe (1 x 108 to 2 x 108 cpm/,lg of
DNA).
D HSV tk Plasmid rescue. Cellular DNA (100 ,ug) was digested with
K
B RNA P 5
ENES
G ..9-------.. TP B ClaI and BamHI. The protocol for plasmid rescue was as
described (2), except that the digested cellular DNA was
mRNA 5' 1
MET STOP used to transform 1.2 ml of competent Escherichia coli
LE392 by the method of Hanahan (13) and that the bacteria
-pBR322 .... M13 _HSV tk O50 bp were concentrated 100-fold. The number of ampicillin-resist-
ant colonies ranged from 2 to 150 per experiment.
RESULTS
E
Overlapping deletion mutations. In our first experiments,
E Na -A Ea s we cotransferred the supercoiled plasmids pBS and pNP into
.--61 I - I
im""'Q mouse Ltk- cells (Fig. 1A). These plasmids contain the 5'
end and the 3' end of the tk gene, respectively, and overlap
FIG. 1. (A, B, and C) Restriction endonuclease maps of plasmids by the 102 bp between the NruI and SphI sites. We refer to
constructed for homologous recombination experiments. (D) Origi- the DNA segment in which a recombination event must
nal 3.5-kbp herpes simplex virus type 1 CL101 BamHI Q fragment occur to generate an intact tk gene as the region of obligate
containing the tk gene. (E) Enlarged map of restriction endonuclease recombination. For this pair of constructions, the region of
fragment from the left-most EcoRV site to the SphI site of tk. The
sizes of the designated restriction endonuclease fragments are obligate recombination is 102 bp in length. We obtained 1.8
determined by the number of nucleotides in the DNA strand after tk+ colonies per plate in hypoxanthine-aminopterin-thymi-
restriction endonuclease digestion. Abbreviations: A, Sacl; B, dine (HAT) medium (Table 1). When we cotransferred
BamHI; C, ClaI; D, NdeI; E, EcoRV; G, BgIII; H, HindIll; K, supercoiled plasmids that overlap by ca. 62 bp, (pNPA35 and
KpnI; N, NruI; P, PvuII; S, SphI; T, BstEII; I, site of insertion of pBS), the frequency fell to 0.3 colonies per plate (Table 1).
heterologous DNA; Y, site of DNA deletion. As a control, each mutant tk plasmid, either supercoiled or
linearized by an appropriate restriction enzyme, was trans-
site of pTK. Plasmid pST14 was constructed by inserting a ferred into Ltk- cells. None of the mutant plasmids pro-
nonphosphorylated 6-bp SmaI linker into the StuI site of duced tk+ colonies in HAT medium.
pST8. Plasmid pST20 was constructed by inserting a We analyzed the DNA from the tk+ cloned transformant
nonphosphorylated 12-bp BamHI linker into the Stul site of cell lines by using Southern blots. The genomic DNA of the
pST8. Plasmid pST24 was constructed by inserting two stable tk+ cell line 161 resulting from the cotransfer of the686 BRENNER, SMIGOCKI, AND CAMERINI-OTERO MOL. CELL. BIOL.
TABLE 1. Number of tk+ colonies with different combinations of increased the frequency of recombination. We found that
mutant plasmidsa cutting pNP at the NruI site before the cotransfer with pBS
Region of Relative increased the number of tk+ colonies from a baseline of 1.8
obligate of tk' per plate to 15 per plate (Table 1).
Mutation Cotransferred plasmids recombi- no.
colonies/ Effect of insertions and deletions. We measured the effect
nation colonies/
(bp) plate on recombination frequency of flanking homology separated
from a 99-bp region of obligate recombination by an inser-
Overlapping pBS x pNP tion of heterologous DNA. Plasmid pST8 was constructed
deletion u u 102 1.8 (6) by inserting an 8-bp StuI linker in the unique NruI site of
u NruI 99 15 (4) pTK (Fig. IB). The linker generates an insertional mutation
SphI NruI 98 22 (1) of 8 bp of heterologous DNA in the coding region of tk.
pBS x pNPA35 62 0.3 (3) Therefore, pBS x pST8 not only has virtually the same
region of obligate recombination as pBS x pNP but also has
598 bp of flanking homologous DNA. The recombination
Insertion pBS X pSTI 99 frequency of pBS x pST8, both uncut, was 63 tk+ colonies
per plate compared with the original pBS x pNP frequency
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u u (I = 8) 63 (12)
u u (I = 14) 56 (4) of 1.8 colonies per plate (Table 1).
u u (I = 20) 56 (2) We next determined the effect of inserting progressively
u u (I = 24) 67 (2) larger fragments of heterologous DNA into the NruI site of
u u (I = 72) 57 (3) tk (the pSTI plasmids, where I, the insertion, ranged from 8
u u (I = 126) 47 (6) to 2,172 bp). In these experiments, the recombination fre-
u u (I = 318) 18 (5)
u u (I = 611) 13 (7) quency showed a progressive decline as the heterologous
u u (I = 2,172) 4.4 (5) insert increased from 8 bp (pST8, giving 63 colonies per
u BamHI (I = 8) 259 (3) plate) to 2,172 bp (pST2172, giving 4.4 colonies per plate)
u NdeI (I = 8) 110 (3) (Table 1).
u BstEII (I = 8) 0 (2) The restriction endonuclease digests of the DNA from
u StuI (I = 8) 183 (7) uncloned and cloned tk+ colonies resulting from the recom-
u BamHI, StuI (I = 8) 33 (2)
u BgIII (I = 8) 206 (9) A B
u BglII (I = 14) 190 (2)
u BglII (I = 20) 225 (2) pNP pBS 161 - 162-
u BglII (I = 24) 47 (4) P P P P H
u BglIl (I = 72) 44 (3) kbp
u BglII (I = 126) 21 (5) --23.1--
u BgIll (I = 318) 17 (5)
u BglII (I = 611) 14 (4)
- 9.4-
u BglII (I = 2,172) 1.9 (3)
---6.6---
Internal pBS x pERV
deletion u
u
u
BglII
59
59
5.5 (3)
35 (2)
.,..
e 44-tW
441
u EcoRV 56 89 (3)
a Mutant plasmids were cotransferred either in the uncut form (u) or after
being cut by the designated restriction endonuclease. tk+ colonies were
counted after 14 days in HAT selective medium. The number in parentheses 7e,r
2.0
signifies the number of plates counted. In the pSTI plasmids, I is the size in bp
of the heterologous DNA that was inserted into the unique NruI site in the -
coding region of tk. The number of tk+ colonies per plate was normalized to
the number of tk+ colonies in the pBS x pST8 plate in each experiment.
plasmids pBS and pNP contained three restriction endonucle- 0.6
ase fragments that hybridized to the tk probe (Fig. 2A, lane
3). Two of the fragments are identical in size to the parental
plasmids linearized with PvuII (Fig. 2A, lanes 1 and 2), a
result consistent with the separate integration of each of
these two plasmids in a head-to-tail array (10). The new
2.1-kbp fragment (Fig. 2A, lane 3) is consistent with an intact 1 2 3 1 2
tk gene resulting from the recombination of the two parental
plasmids within the region of obligate recombination. FIG. 2. Southern blots of plasmid DNA and cellular DNA from
Figure 2B shows a similar result for another tk+ cell line tk+ cell lines derived from cotransfer of pNP and pBS into Ltk-
(line 162) resulting from the recombination of pBS and pNP. cells and probed with the 3.5-kbp BamHI fragment containing the tk
The new restriction endonuclease fragments are the 2.1-kbp gene. (A) Lanes 1, 2, and 3 show PvuII digests of the DNAs of pNP,
PvuII fragment (Fig. 2B, lane 1) and the 4.0-kbp HindIII pBS, and cloned line 161, respectively. The asterisk (*) denotes a
new 2.1-kbp restriction endonuclease fragment predicted from re-
fragment (lane 2) predicted for the recombination product combination (Fig. 1). (B) Lane 1, cloned line 162 digested with PvuII
(Fig. 1A). with the asterisk (*) showing the new 2.1-kbp fragment; lane 2,
We next determined that cutting one of the plasmids with cloned line 162 digested with HindIll with the asterisk (*) showing
a restriction endonuclease within the region of homology the new 4.0-kbp fragment predicted by recombination.VOL. 5, 1985 HOMOLOGOUS RECOMBINATION IN MOUSE L CELLS 687
bination of pBS and pST126 (a 126-bp insertion), both uncut, 43
were analyzed with tk as the probe (Fig. 3). In all three lanes, U)
there is a 2.8-kbp restriction enzyme fragment resulting from U
z
a ClaI and BstEII digestion (Fig. 3, lanes 2, 4, and 6); we a 3 _
would expect such a fragment from a recombinant tk gene 0
that had exchanged flanking parental markers (Fig. 1B). A z
gene conversion or double-reciprocal exchange might re-
move the insertion in pST126, creating an intact tk gene with 2
0.,
a new 800-bp restriction enzyme fragment when digested 0 m
with NdeI and NruI. Such a fragment was not present in
digests of the genomic DNA (Fig. 3, lanes 3, 5, and 7). I- 1 .
.
The effect of linearizing one of the pSTI insertion plasmids 0
by cutting to the left of the recombination region before
cotransfer with supercoiled pBS varied markedly. Cutting
pST8 with restriction endonucleases to the left of the region 0 50 100 150 200 250 300 350 400 450 500 550 600 21502200
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of obligate recombination with NdeI, BamHI, BglII or StuI INSERTION SIZE (bp)
(all cutting at unique sites) before the cotransfer with uncut
pBS increased the frequency approximately two- to fourfold FIG. 4. The ratio of transformation frequencies for each pSTI
(Fig. 1B and Table 1). The effect is smallest for the double- plasmid (when the insertion size I is 8, 14, 20, 24, 72, 126, 318, 611,
strand break introduced by NdeI, probably because the or 2,172 bp) was derived from the relative number of tk+ colonies
NdeI site is outside of the region of flanking homology. In per plate when BglII-cut pSTI was cotransferred with pBS divided
by the number when uncut pSTI was cotransferred with pBS. The
contrast to the effect of introducing a cut to the left of the number of tk+ colonies per plate resulting from the cotransfer of a
region of obligate recombination, cutting pST8 to the right of pSTI plasmid with pBS into Ltk- cells is described in Table 1.
this region (with BstEII) decreased the recombination fre-
quency to zero. ting pST24, pST72, pST126, pST318, pST611, and pST2172
Cutting pST14 and pST20 to the left of this region with at the BglII site failed to increase the recombination fre-
BglII before cotransfer with supercoiled pBS increased the quency (Table 1 and Fig. 4).
recombination frequency three- to fourfold. However, cut- The plasmid pERV has a 104-bp deletion in tk, a 59-bp
region of obligate recombination with pBS, and 537 bp of
flanking homology (Fig. 1C). When pERV and pBS are
(0
N aligned with respect to their regions of homology, the
equivalent of a 104-bp insertion is created in pBS (Fig. 1).
uncl 178 179 The recombination frequency of pBS and pERV, both
uncut, was 5.5 colonies per plate. When pERV was cut at its
T C,T D,N C,T D,N C,T D,N unique BglIH site before cotransfer with pBS, the recombi-
kbp nation frequency increased sixfold (Table 1).
23.1- We determined that double-strand gaps in our plasmids
9.4- are recombinogenic in L cells, as has been previously
6.6- demonstrated in yeast cells (22, 23). Cutting pERV with the
restriction endonuclease EcoRV created a 104-bp double-
4.4- strand gap in tk. When plasmid pERV was cut with EcoRV
before cotransfer with pBS, the recombination frequency
* 4 - * a w - A increased 300-fold compared with that of the uncut pNPA35
2.3- plasmid (these two plasmids have almost identical regions of
2.0- obligate recombination, but pNPA35 lacks flanking homol-
ogy).
Plasmid rescue. We cloned products of homologous recom-
bination by the technique of plasmid rescue (1, 2, 24). For
this purpose, genomic DNA from a cloned tk+ transformed
cell line resulting from the recombination of uncut pBS and
0.6- uncut pST8 (line 175) was cut with BamHI and ClaI. Two
plasmids, p175B1 (Fig. 5) and p175B2, were rescued in this
experiment. The restriction endonuclease map of p175B2
was identical to the parental plasmid pST8, including the
StuI site.
1 2 3 4 5 6 7
FIG. 3. Southern blot of plasmid pST126 DNA and cellular DNA B A K P G NS T P DI P B
from tk+ cell lines derived from cotransfer of pST126 and pBS, both i ...I
uncut, into Ltk- cells and probed with tk. Lane 1, Plasmid pST126 Ap
linearized with BstEII. Forty uncloned pooled colonies (uncl) and
cloned lines 178 and 179 were digested with either ClaI and BstEII
(C, T) or NdeI and NruI (D, N). The asterisk (*) shows the new pBR 322 . M13 _HSVtk JV'\,Cel1ular
1 Kb
|-1
2.8-kbp restriction endonuclease fragment predicted from recombi-
nation with exchange of ClaI and BstEII flanking markers (see Fig. FIG. 5. Restriction endonuclease maps of plasmid p175B1 res-
1). cued from cloned tk+ cell line 175. Abbreviations are as in Fig. 1.688 BRENNER, SMIGOCKI, AND CAMERINI-OTERO MOL. CELL. BIOL.
p175B1 is a product of recombination of the parental cloned DNAs from tk+ transformant cell lines by plasmid
plasmids. The 5' end of its tk gene including the 5' flanking rescue. Plasmid p175B1 contains a functional tk gene and is
restriction endonuclease site KpnI came from the parental the product of recombination of DNA sequences from the
plasmid pBS, and the 3' end of its tk gene including the 3' two mutant plasmids. Since the DNA sequences flanking the
flanking restriction endonuclease site BstEII and vector 5' end of the tk gene (in pBS) have been lost, the tk gene
DNA came from pST8. In addition, there is a 500-bp contained in this plasmid is not flanked by the predicted
fragment containing a new SacI site that is absent in both restriction endonuclease sites.
parental plasmids. The 500-bp fragment was used as a probe Length of overlapping DNA sufficient for recombination.
in a Southern blot of mouse Ltk- genomic DNA. A blur or Previous studies with cotransferred mutant plasmids used a
smear pattern was observed (data not shown). This result is range of overlapping homologous DNA from 3 kbp in an
consistent with the presence of a mouse chromosomal intermolecular recombinant event (5) down to 14 bp in an
repetitive sequence within the probing fragment. The origin intramolecular recombinant event (a single plasmid contain-
of this sequence is either the carrier DNA or the chromo- ing two different overlapping mutants [29]). Our data are
somal insertion site. consistent with these reports and demonstrate that 62 bp of
We confirmed that the plasmid p175B1 was not rearranged overlapping homology is sufficient for the intermolecular
during its rescue in E. coli. We probed a Southern blot of the recombination of tk mutants.
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genomic DNA of the cloned line 175 with tk (data not Evidence for homologous pairing in recombination. In
shown). The following fragments were found in both the principle, there are multiple ways in which two mutant DNA
rescued plasmid p175B1 and the genomic DNA: the 2.8-kbp molecules sharing homology can be joined to produce a
BamHI-BstEII fragment, the 2.5-kbp SacI-BstEII fragment, wild-type gene (i.e., homologous recombination). In the
and the 2.3-kbp KpnI-BstEII fragment. When pl75B1 was break-and-join model (37), double-strand cuts in the DNA
transferred into Ltk- cells, it produced the same number of are introduced and followed by ligation of the double-strand
tk+ (HAT-resistant) colonies as did a plasmid containing the ends. Since there are few data available from studies of
wild-type tk gene (pTK). Therefore, the rescued plasmid mammalian cells to support this model, we shall not discuss
pl75B1 contains a functional gene. it further. A second model for which there are recent
supporting data from Lin et al. (18) is that invoking single-
DISCUSSION strand annealing. In this model the creation of double-strand
breaks is followed by single-strand exonuclease digestion of
In the following sections we discuss how our data show both mutant genes by a 3' or 5' exonuclease, complementary
the following. (i) The tk+ phenotype is the result of recom- single-strand annealing, and ligation to form a wild-type
bination of the DNA of the two cotransferred mutations and molecule.
not due to complementation at the protein level. (ii) The Extensive data from E. coli and fungi support a third
recombination events are akin to those observed in E. coli model in which there are at least two distinguishable steps:
and yeast cells in that they are the result of homologous first, an alignment of the two DNAs in which at least one of
pairing followed by heteroduplex formation and extension. the substrates is double stranded (a process that in reactions
These data suggest that these processes rather than ligation of recA in vitro is called homologous pairing [27]), and a
or single-strand annealing or both are responsible for the second step, initiated by either single-strand nicks (19, 26,
recombination events observed in mammalian somatic cells 27) or double-strand breaks (40) and leading to heteroduplex
in culture. (iii) Insertions smaller than 24 bp are handled formation and extension. A crucial difference between the
differently by the recombination process than inserts larger third and second model is that in the third model, the
than 24 bp. (iv) Insertions and deletions are handled differ- substrate DNAs interact with each other before a het-
ently by the recombination machinery, that is, there is a eroduplex is formed. Thus, in the third model, homologous
donor-recipient asymmetry. DNA separated by a double-strand break or gap from the
Homologous recombination of plasmid DNA results in the region of obligate recombination can be involved in the
tk+ phenotype. Previous experiments demonstrated that initial pairing of the two substrates. As we will discuss
when two complementary nonleaky mutant genes were shortly, this interaction can easily account for the beneficial
cotransferred into mammalian somatic cells, some of the effect of flanking homology on the recombination frequency.
cells expressed the wild-type phenotype (5, 18, 21, 25, 32, Our data from experiments with insertions, deletions, and
35, 38). In these studies, Southern blots were used as the double-strand gaps support the concept that homologous
main evidence that this change of phenotype represented pairing of the mutant plasmids occurs in recombination. In
homologous recombination of the cotransferred DNA se- particular, the double-strand break repair model, a version
quences; i.e., if a new restriction fragment was the predicted of the third model just discussed (40), explains many of our
length, then it was assumed to represent the recombinant results with mutant plasmids containing double-strand breaks
functional gene. However, it is difficult to extrapolate from and double-strand gaps. In this model, a homologous recom-
Southern blots of DNAs from transformed cell lines to bination event is initiated by a double-strand break which is
molecular mechanisms. enlarged to a double-strand gap by exonuclease activity. The
Since multiple copies of both parental plasmids integrate gap is subsequently repaired by DNA synthesis, using a
into transfected cells (35) (Fig. 2 and 3), the tk+ phenotype displaced single-strand DNA as a template. For example, we
might result from interallelic complementation at the protein can compare pBS cotransferred with pNP cut with NruI,
level, as has been previously demonstrated in E. coli, and pST8 cut with StuI plus BamHI, or pST8 cut with StuI.
not from the recombination of DNA sequences. Also, the Plasmid pNP cut with NruI and pST8 cut with StuI plus
DNAs of several of the cloned tk+ colonies produce compli- BamHI have the same region of obligate recombination
cated Southern blots with many nonparental restriction without any flanking homology (Fig. 1A and B). The 6- to
endonuclease fragments, some of which mimic the fragments 10-fold increase in the frequency of recombination obtained
expected for recombinants. To prove that recombination of by cutting pST8 with StuI relative to the other two sub-
DNA sequences was responsible for the tk+ phenotype, we strates demonstrates the benefit of flanking homology sepa-VOL. 5, 1985 HOMOLOGOUS RECOMBINATION IN MOUSE L CELLS 689
rated from the region of obligate recombination by a double-
strand break (Table 1). pBS
The double-strand break repair model also explains the
recombinogenic effect of the flanking homology separated
from the region of recombination by a double-strand gap. 5' E N
When pERV cut with EcoRV (which creates a double-strand 6 I I
gap separating the region of obligate recombination from 540
bp of flanking homology) is cotransferred with uncut pBS,
the recombination frequency is sixfold higher than when 3' L R
pNP without flanking homology is cut with NruI and co-
transferred with pBS. Recently, Kucherlapati et al. (14) have
also demonstrated the recombinogenic effect of double-
strand gaps by studying the transfer of plasmids containing
mutations in the bacterial neo gene into the EJ human
II E E S\
bladder carcinoma line.
pERt
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In contrast to a model invoking homologous pairing, the
proposed version of the single-strand annealing model (ref-
erence 18, discussed above) cannot easily explain the recom- FIG. 6. Homologous pairing of pBS with pERV. Plasmid pERV
binogenic effect of flanking homology separated from the was digested with EcoRV to create a double-strand gap.
region of obligate recombination by a double-strand break or
gap. An example is the experiment in which pBS is cotrans- heteroduplex formation, the heteroduplex can only easily
ferred with pERV cut with EcoRV, a double-strand gap extend through at most a 20-bp insertion of heterologous
molecule (Fig. 6). If a 5' exonuclease digests the right arm of DNA on the initiating DNA (pST20 cut with BglII x pBS).
linearized pERV (R), then the 3' right arm is exposed for That is, homologous pairing is distinguishable from subse-
annealing with the region between E and S on the comple- quent heteroduplex formation and extension.
mentary top single strand of pBS. This strand of pBS has The ability of E. coli recA to extend heteroduplexes
become exposed as the result of the combined action of an through a heterology in vitro has been studied by others, but
endogenous endonuclease cutting to the right of the right- the results are controversial. They indicate that either only
most E and the same 5' exonuclease. However, the same 5' inserts smaller than 4 bp (42) or inserts of several hundred bp
exonuclease activity digesting the left arm of pERV (L) will (3) can be accommodated in the heteroduplex. One interpre-
destroy the pERV strand complementary to the top strand of tation of our data is that this value might lie between 20 and
pBS. Since a given exonuclease that acts only on the 3' end 24 bp in L cells.
or the 5' end would expose different strands on the ends Another interpretation of these data is that the discon-
flanking a break, only one of the DNAs surrounding the tinuity in the recombination frequency for different insert
break can anneal with the other mutant gene. Therefore, the sizes is a reflection of the mismatch repair machinery of
homology on the other side of the break or gap cannot these cells. Thus, the heteroduplex procedes through hetero-
participate in the recombination process. logous inserts greater than 24 bp, but the cells can only
Effect of heterologous inserts on homologous recombination. repair less than 24 bp of mismatched DNA. Although we
The effect of heterologous inserts was studied by cotransfer- cannot distinguish between these two possibilities, in either
ring pBS with plasmids similar to pST8 but with the addi- case we are separating a later step in a recombination event
tional flanking homology separated by larger heterologous from the earlier homologous pairing.
insertions (the pSTI plasmids) or a 104-bp deletion (pERV). Furthermore, our data demonstrate an asymmetry in how
When the two plasmids were cotransferred in the uncut heterologous inserts are handled by a heteroduplex. The
form, the frequency of recombination was significantly higher heteroduplex can more easily accommodate an insertion
relative to the pBS x pNP experiment (in which pNP has no larger than 20 bp on the noninitiating DNA (pERV cut with
additional flanking homology), despite insertions as large as BglII x pBS) to generate an intact tk gene. The reason for
611 bp (pST611) and a 104-bp deletion (pERV) (Table 1). As this asymmetry in the handling of insertions on the two
the insertion increased, there was a decline in the frequency DNAs is not clear. A less dramatic asymmetry has been
of recombination. reported for mismatched heteroduplexes made by purified E.
The effect of linearizing plasmids in the pSTI series or coli recA protein in vitro (3). In those experiments, however,
pERV by cutting to the left of the insertion or deletion at the the inserts in the initiating (single-strand) DNA were more
unique BglII site (G in Fig. 1) before their cotransfer with efficiently accommodated than the inserts in the non-
supercoiled pBS plasmid varied markedly (Table 1). In initiating (double-strand) DNA.
particular, cutting at the BglII site on an insertion mutant of The fact that only a heterologous insert on the initiating
-20 bp or at the 104-bp deletion mutant increased the DNA of less than 24 bp can be easily accommodated in the
frequency of recombination three- to fourfold for these putative heteroduplex might be related to an observation
insertions and sixfold for the deletion. However, cutting at that we made when examining the recombinant joints be-
the same BglII site in an insertion mutant of .24 bp did not tween two nonhomologous DNAs sharing an area of partial
increase the frequency (Fig. 4). homology (1). We proposed that when the two DNAs were
The recombination process appears to be dissectable into aligned at the areas of partial homology, one molecule
at least two phases. The benefit of flanking homology seen in donated an end from within the area of homology and the
crosses with uncut mutant plasmids probably reflects the other donated an end also from within that area or from the
fact that the initial homologous pairing of sequences or flanking area of heterology. If the origin of one of the ends
synapse formation can accommodate a large heterologous was at least 24 base pairs away from the border of shared
fragment (pST611). If we assume that the creation of a homology (or the putative heteroduplex), a "filler" of 19
double-strand break (the BglII cut) serves as the site of base pairs of a third DNA was inserted between the two690 BRENNER, SMIGOCKI, AND CAMERINI-OTERO MOL. CELL. BIOL.
ends. If the origins of both ends were within the area of meiotic gene conversion, or "wanderings of a foreign strand,"
partial homology or if the origin of one end was less than 24 p. 289-339. In J. N. Strathern, E. W. Jones, and J. R. Broach
bp from the border of shared homology, both ends were (ed.), The molecular biology of the yeast Saccharomyces, vol.
joined directly to each other. We speculate that these 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
10. Folger, K. R., E. A. Wong, G. Wahl, and M. R. Capecchi. 1982.
experiments are formally very similar to those presented Patterns of integration of DNA microinjected into cultured
here. In both cases there is a discontinuity in the results of mammalian cells: evidence for homologous recombination be-
the recombination when the inserts are smaller than 24 bp tween injected plasmid DNA molecules. Mol. Cell. Biol.
compared with when the inserts are larger. 2:1372-1387.
The role of palindromes. We note that in the pSTI series, 11. Gutai, M. W. 1981. Recombination of SV40-infected cells:
all the inserts up to 24 bp in length are perfect palindromes nucleotide sequences at viral-viral recombinant joints in natu-
up to 30 bp in length (24 bp plus the 3 bp on both ends from rally arising variants. Virology 109:344-352.
the original NruI site). The increased frequency of recombi- 12. Gutai, M. W. 1981. Recombination of SV40-infected cells: viral
nation when these plasmids are crossed with pBS compared DNA sequences at sites of circularization of transfecting linear
DNA. Virology 109:353-365.
to a pNP x pBS experiment is not attributable to these 13. Hanahan, D. 1983. Studies on transformation of Escherichia coli
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able palindromes also shows a similar increase in frequency. 14. Kucherlapati, R. S., E. M. Eves, K. Song, B. S. Morse, and 0.
In fact, these data demonstrate that in our experimental Smithies. 1984. Homologous recombination between plasmids in
system small palindromes do not play a significant role in mammalian cells can be enhanced by treatment of input DNA.
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Conclusion. Mammalian somatic cells are capable of me- 15. Laban, A., and A. Cohen. 1981. Interplasmidic and intraplas-
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ACKNOWLEDGMENTS shotgun DNA sequencing. Nucleic Acids Res. 9:310-321.
We thank F. Lin and N. Sternberg for allowing us to read their 21. Miller, C. K., and H. M. Temin. 1983. High-efficiency ligation
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Hsieh, and S. Kato for valuable advice and discussion and J. Glass, Science 220:606-609.
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