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(Received for publication, May 15, 1995; and in revised form, August 2, 1995) From the
To test the influence of cytosine methylation on homologous
recombination and the rejoining of DNA double strand breaks in
mammalian cells, we developed a sensitive and quantitative assay system
using extrachromosomal substrates. First, methylation was introduced
into substrates in vitro with the prokaryotic SssI
methylase, which specifically methylates the C-5 position of cytosine
bases within CpG dinucleotides, mimicking the mammalian DNA
methyltransferase. Next, methylated substrates were incubated in
mammalian cells for a sufficient length of time to recombine or rejoin
prior to substrate recovery. Results from bacterial transformation of
the substrates and from direct Southern analysis demonstrate that
cytosine methylation has no detectable effect on either DNA end-joining
or homologous recombination. Thus, the components of the protein
machinery involved in these complex processes are unaffected by the
major DNA modification in mammalian cells. These results leave open the
possibility that methylation may modulate the accessibility of these
components to chromosomal DNA by altering local chromatin structure.
Two key processes in the maintenance of genomic integrity in
mammalian cells are DNA end-joining, a nonhomologous process in which
DNA breaks are rejoined, and homologous recombination. Both of these
processes are mobilized in the repair of double strand breaks in
chromosomal DNA (1) and in transfected extrachromosomal DNA
(reviewed in (2) and (3) ). Homologous recombination
of transfected DNA substrates is postulated to occur primarily by the
nonconservative single strand annealing
pathway(4, 5) . In this pathway, the two substrates
contain double strand breaks at or near their homologous regions on
which an exonuclease (or helicase) acts to produce single strands. The
homologous single strands anneal and subsequent processing steps result
in the completion of the recombination event. DNA end-joining has
also been extensively studied by transfection of substrates into
mammalian cells(2) . Surprisingly, many different combinations
of DNA ends can be rejoined efficiently in vivo. In addition
to the precise ligation of compatible ends, noncompatible ends are also
rejoined. For example, blunt-ended DNA ends can be rejoined to either
3` or 5` overhangs. The rejoining of the noncompatible ends frequently
occurs within very short homologies near the DNA ends(2) .
Mechanistically, this type of end-joining may be similar to the single
strand annealing pathway of homologous recombination, the key
distinction being the length of the homology. Although the protein
machinery involved in either recombination or DNA end-joining is not
well characterized, a number of components, including exo- and
endonucleases, DNA polymerase, ligase, and strand annealing proteins,
can be expected to participate. The major base modification of
mammalian DNA, cytosine methylation, occurs on the C-5 position of
cytosines within the context of CpG dinucleotides (reviewed in (6) ). Cytosine methylation has several demonstrated
biophysical consequences on DNA and, thus, it may modulate the
enzymatic activity of some of the components involved in recombination
or end-joining. For example, methylation increases the melting
temperature of naked DNA(7) . It also has other effects, such
as influencing the extrusion of DNA cruciforms(8) , as well as
the transition of DNA from the B form to Z form(9) . However,
it appears to have almost no influence on the intrinsic flexibility of
DNA(10) . Functionally, methylation has a critical role in
transcriptional regulation, and it has been implicated in both the
establishment and maintenance of X chromosome inactivation and genomic
imprinting patterns(6) . The consequences of cytosine
methylation in gene regulation may be the result of directly or
indirectly altering the binding or activity of transcription factors
and chromatin proteins (11) . Although Saccharomyces
cerevisiae, Caenorhabditis elegans, and Drosophila have no detectable methylation, the importance of methylation in
mammals has been underscored by gene knockout experiments in which it
was found that mice defective in the DNA methyltransferase die during
embryonic development(12) . Methylation may also play a role in
modulating the timing of replication. For example, the inactive X
chromosome is replicated later than the active X chromosome in female
cells(13, 14) . At present, it is unknown whether
methylation affects homologous recombination in either mitotic or
meiotic mammalian cells. However, it has been observed that
recombination rates differ at identical chromosomal regions between
males and females. Overall, females have a higher rate of recombination
than males, although some chromosomal regions recombine more frequently
in males than in females(15) . Methylation patterns also differ
between male and female gametes, with spermatogenic cells having an
overall higher level of methylation than oogenic
cells(16, 17) . Thus, methylation could potentially
directly or indirectly suppress recombination rates. Related to this,
it has been demonstrated that methylation decreases the site-specific
recombination of antigen receptor genes, at least in some contexts. In
this report, we have begun to address whether methylation has a direct
affect on either homologous recombination or DNA end-joining in
mammalian cells.
Recombination
substrates were constructed based on the parental Mneo plasmid. Mneo
has an intact Tn5 neo gene and confers kanamycin resistance
(Kan
Figure 1:
Structure of
the recombination substrates. The parental plasmid Mneo contains a
functional Tn5neo gene, whereas the recombination substrates M5neo and
M3neo contain 3`- and 5`-truncated neo genes, respectively.
Base pair positions of relevant restriction sites are indicated for
Mneo.
The
experimental design is shown in Fig. 2. M5neo/S and M3neo/AP are
methylated in vitro with a cytosine methylase and then
electroporated into mammalian cells. Cells are incubated for 4 h, after
which time plasmid DNA is recovered. We chose 4 h, since previous
results have shown that recovery of recombined DNA is optimal at this
time point. (
Figure 2:
Experimental design to determine the
effect of CpG methylation on extrachromosomal homologous recombination
in mammalian cells. M5neo/S and M3neo/AP can recombine within the
352-bp neo gene homology region (black shading) to generate a
functional neo gene. Recombination can also occur downstream of the neo gene, as indicated. The downstream homology (thick
line) is within pUC19 sequences and is 487 bp. EP,
electroporation.
Figure 3:
Agarose gel electrophoresis to demonstrate
the resistance of in vitro methylated DNA to HpaII
digestion. C
Prior to transfection of DNA into mammalian cells, we monitored the
transformation efficiency of the methylated plasmid DNAs in E.
coli. DNA was electroporated into E. coli strain DH12S,
which is defective in the restriction of methylcytosine containing DNA.
Transformation of the parental Mneo plasmid resulted in almost 2
Transformation of M5neo/S,
either methylated or unmethylated, resulted in an approximately
500-fold reduction in the number of Amp The
other recombination substrate, M3neo/AP, transforms bacteria to
Amp M5neo/S and M3neo/AP were also mixed prior to
transformation into bacteria. Although Amp
Figure 4:
Southern blot analysis of DNA recovered
from transfected COS1 cells. DNA was methylated in vitro (indicated by C
Retention of
methylation was monitored by HpaII digestion. Substrates that
were methylated in vitro retained their methylation, as
evidenced by their resistance to HpaII (Fig. 4). By
contrast, unmethylated substrates were completely digested by HpaII to smaller fragments.
No Kan The number of Amp The DNA that was transfected into COS1 cells was first
cleaved by the restriction endonucleases and then methylated. Since
some of the CpG dinucleotides are located within or adjacent to the
restriction sites, it is possible that these sites may remain
unmethylated. To rule this out, we also performed experiments in which
the methylation was performed prior to the restriction digestion.
Nearly identical results were obtained in this experiment to those
shown in Table 2(data not shown), proving that the methylation
at the ends does not reduce end-joining efficiencies. As mentioned
above, the difference between the M5neo/S and M3neo/AP plasmids in
end-joining efficiencies in bacteria is likely due to the close
proximity of the AatII site to the amp gene in M3neo.
Alternatively, the particular restriction enzyme cleavage for the two
plasmids may play a role. However, following COS1 cell transfection
there is little difference between the two plasmids in the generation
of Amp Individual recombination and end-joining products were
analyzed by preparing plasmid DNA from bacterial colonies and
subjecting them to restriction analysis. For the recombination
products, plasmids were prepared from 30 Kan
Figure 5:
Structure of recombination and end-joining
products. DNA recovered from cotransfection of COS1 cells with M5neo/S
and M3neo/AP was electroporated into bacteria. Plasmid DNA was prepared
from either Kan
To
examine end-joining products, plasmids were prepared from 10
Kan
Figure 6:
Southern blot analysis of DNA recovered
from transfected COS1 cells. A, structure of recombination and
end-joining products, as described in the text. Both intermolecular (R, RE, ER, EE, and E) and intramolecular (3E1,
3E2, and 5E) products are shown. Only intermolecular
products which have recircularized are diagrammed. B, Southern
analysis. DNA was cleaved with HindIII/BamHI prior to
gel electrophoresis. The positions of these sites are indicated in Fig. 1. T lanes: DNA recovered from transfected COS1
cells; C lanes: DNA prior to transfection into COS1 cells. The HindIII/BamHI neo fragment was used as the
probe.
For
Southern analysis, DNA is cleaved with HindIII and BamHI (Fig. 1) and probed with a neo gene
fragment (Fig. 6B). No recombination or end-joining
products are detected without prior transfection of DNA into mammalian
cells (Fig. 6B, ``C'' lanes). However, a variety of products are detected upon
transfection of either the methylated or unmethylated DNAs into COS1
cells (Fig. 6B, ``T'' lanes). Cotransfection of M5neo/S and M3neo/AP results in
formation of both recombination and end-joining products. Recombination
occurred within the neo gene (R and RE products) for about 5-10% of the input substrates and is
similar in both the methylated and unmethylated samples. The Southern
assay gives an apparently higher level of recombination than does the
bacterial transformation assay. This difference is due to the nature of
the two assays. Southern analysis directly examines the product of
recombination within the neo gene. Bacterial transformation to
kanamycin resistance requires a second event (recombination or
end-joining) within the plasmid sequences, in addition to recombination
within the neo gene, so as to generate a closed circular
product. This requirement for a second event lowers the measured level
of recombination in the bacterial transformation assay. As with the
recombination product, end-joining products are also detected at
similar levels in both the methylated and unmethylated samples. The
intermolecular products (ER/EE and E) are only
detected upon cotransfection of M5neo/S and M3neo/AP, whereas the
intramolecular product 3E1 is detected in both the cotransfection and
the transfection of M3neo/AP alone. The intramolecular product derived
from M5neo/S is not separated from the input linearized M5neo/S in the HindIII/BamHI digest shown in Fig. 6B. However, this product is detected using other
restriction digests (data not shown). We demonstrate that mammalian cytosine methylation has little
or no effect on extrachromosomal recombination in tissue culture cells.
End-joining processes also are unaffected by CpG methylation. These
results were obtained using a highly sensitive bacterial transformation
assay and were confirmed by direct Southern analysis of DNA recovered
from transfected COS1 cells. These results are not unique to COS1
cells, since experiments performed in mouse embryonic stem cells have
yielded similar results. ( Our substrates consist of bacterial
plasmids carrying portions of the Tn5 neo gene. As with most
bacterial genes, Tn5 neo is G/C-rich, rendering it a good
model system in which to examine the effect of cytosine methylation.
The CpG content is 9% of dinucleotides in the entire neo gene
as well as 9% of dinucleotides in the 352 bp neo homology
region. Even though the CpG content in mammalian genomic DNA is in
general much lower (e.g. 1% of dinucleotides in human DNA),
CpG islands have a higher CpG content. For example, 14% of
dinucleotides in the 5` region of the human PGK1 gene are
CpG(24) . Therefore, it is reasonable to use Tn5 neo as a model system to study the effect of cytosine methylation on
homologous recombination in mammalian cells. As with
extrachromosomal recombination, no effect of cytosine methylation is
detected on DNA end-joining frequencies or products. CpG dinucleotides
are located at two of the ends, as well as further in from the ends (Fig. 7). For M3neo/AP, there is a CpG dinucleotide within the AatII overhang itself, as well as 15-bp upstream of the
cleavage site. The PstI end of M3neo/AP also has a CpG
dinucleotide located 15 bp upstream. For M5neo/S, there are two CpG
dinucleotides directly adjacent to the SphI site on one end.
Both ends also have other CpG dinucleotides located further upstream (Fig. 7). The major end-joining products detected by the
bacterial transformation assay involves recircularization of the
M3neo/AP and M5neo/S plasmids (3E2 and 5E). As shown in Table 2,
they are not affected by methylation. Thus, methylation within an
overhang or adjacent to an overhang does not interfere with
end-joining. We cannot rule out, however, that there are subtle
differences between the methylated and unmethylated end-joining
products from the M3neo/AP transformation. However, the M5neo/S
end-joining appears to be identical whether or not the substrate is
methylated, since the SphI cleavage site is restored. Thus,
end-joining appears unaffected by general methylation of the substrates
or methylation directly at the ends of a broken DNA molecule. The
biochemical details of end-joining have not been fully elucidated.
However, proteins such as the Ku autoantigen and its associated
DNA-activated protein kinase are likely involved, since this complex
binds to DNA ends(25) . It may be that methylation does not
affect binding of this complex or subsequent processing and ligation
steps postulated to be involved in end-joining.
Figure 7:
Sequences at the ends of the end-joining
substrates. The methylated residues are indicated by an asterisk. For M3neo/AP, only the two ends that form in the
intramolecular product 3E2 are shown.
Our results have
demonstrated that extrachromosomal homologous recombination is
unaffected by CpG methylation. Recombination in these substrates likely
occurs via the single strand annealing pathway(4, 5) .
This pathway has the requirement that both recombination substrates be
cut at or near the regions of homology(26) . The DNA ends may
then provide an entry site for an exonuclease such that single strands
are exposed for annealing. Mechanistically, our results suggest that
CpG methylation has little or no effect on the exonuclease and strand
annealing activities. A second pathway for homologous recombination,
double strand break repair (27) has also been shown to be
operational in mammalian cells(28, 29, 30) .
In this pathway, a DNA substrate containing a double strand break is
repaired from an unbroken homologous DNA template after strand
invasion, conserving both partners of the recombination event. We
cannot rule out that CpG methylation may have an effect on this
pathway. Experiments that we have performed in which only one of the
recombination substrates is broken and the other is introduced as a
supercoiled plasmid have shown no difference between methylated and
unmethylated substrates. Previous work has shown that CpG
methylation and the accessibility of loci to site-specific recombinases
are interrelated. A transgene locus that can undergo V(D)J
recombination has been identified that is refractory to recombination
when methylated yet accessible for recombination when
unmethylated(32) . CpG methylated minichromosomes are also
inaccessible for V(D)J recombination(33) . Since the inhibition
of V(D)J recombination is apparent only after replication of the
minichromosomes, the interpretation of these results is that CpG
methylation does not directly affect recombination, but rather results
in an altered chromatin structure upon replication which inhibits V(D)J
recombination(33) . In addition, demethylation of endogenous T
cell receptor and immunoglobulin gene loci has been shown to occur upon
activation of lymphocytes which leads to T cell receptor recombination
and Ig class switching, another site-specific recombination
event(34) . Thus, although we find that methylation has no
effect on the homologous recombination of extrachromosomal DNA,
methylation may yet to be found to exert effects on chromosomal DNA by
altering the accessibility of DNA to recombination machinery.
Volume 270,
Number 40,
Issue of October 06, pp. 23838-23844, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Plasmid Constructions and Methylation
Reactions
DNA manipulations were performed according to standard
procedures(18) . The Tn5 neo gene fragments were
derived from plasmid pSV2neoM7. Plasmid pSV2neoM7 has a hybrid neo gene in which the 5` portion of the gene is derived from pSV2neo (19) and the 3` portion of the gene is derived from
pMC1neopA2(20) . The fragments that gave rise to pSV2neoM7 are
the 961-bp (
)NdeI/EagI from pSV2neo, the
816 bp EagI/BamHI fragment from pMC1neopA2, and PstI/BamHI cleaved pUC19. The PstI and NdeI sites were blunted prior to ligation by treatment with T4
DNA polymerase and Klenow fragment, respectively. Plasmid Mneo was
constructed by cloning the 1206-bp HindIII/BamHI Tn5 neo gene fragment from pSV2neoM7 into HindIII/BamHI-cleaved pUC19. The recombination
substrate M5neo was constructed by inserting the 885-bp HindIII/SphI Tn5 neo gene fragment of
pSV2neoM7 into HindIII/SphI-digested pUC19. Plasmid
M3neo was generated by ligating the 673-bp PstI/BamHI neo gene fragment of pSV2neoM7 into PstI/BamHI-digested pUC19. The latter two plasmids
have 352 bp of homology between the PstI and SphI
sites of the neo gene. Prior to transfection, M5neo was
cleaved with SphI, and M3neo was cleaved with PstI
and AatII. Cleaved DNA was methylated in vitro via
the SssI methylase according to the manufacturer's
instructions (New England Biolabs). The samples of unmethylated DNAs
were incubated in methylation buffer without the SssI
methylase, as a control.Bacterial Transformation
Escherichia coli DH12S, genotype araD139 
(ara, leu)7697
lacX74 galU galK mcrA
(mrr-hsdRMS-mcrBC) rpsL deoR Ø80dlacZM15 nupG recA1/F`proABlacI
ZM15, was prepared for transformation via electroporation as
recommended by the manufacturer (Life Technologies, Inc.). Bacteria (40
µl) were mixed with 0.5 µl of DNA and electroporated in a
0.1-cm cuvette using a Bio-Rad gene pulser with a setting of 1.8 kV.
They were then placed in 1 ml of SOC medium for a 1-h incubation at 37
°C prior to plating on ampicillin- or kanamycin-containing plates.Cell Culture and DNA Recovery
COS1 cells were
cultured in 150-cm
tissue culture flasks and transfected
via electroporation. Cells were harvested at subconfluence and
resuspended in phosphate-buffered saline at a concentration of 10
cells/ml. A volume of 0.8 ml of cells was placed in a 0.4-cm
cuvette and electroporated using a Bio-Rad gene pulser with a setting
of 250 V, 960 µF. Just prior to electroporation, 30 µg of each neo plasmid DNA was mixed with the cells. Cells were washed
twice in phosphate-buffered saline immediately after electroporation to
remove untransfected DNA and then incubated in tissue culture medium at
37 °C. To recover transfected DNA, cells were harvested after 4-h
incubation. They were trypsinized and then washed three times in
phosphate-buffered saline to remove any remaining untransfected DNA.
Transfected DNA was recovered as described previously (21) and
resuspended in 50 µl of HO. Southern analysis was
performed using 1-2 µl of DNA according to standard
procedures(18) .
A Sensitive Assay for Extrachromosomal Homologous
Recombination
To determine whether cytosine methylation
influences the efficiency of extrachromosomal homologous recombination
in mammalian cells, we developed a sensitive and quantitative assay
based on bacterial transformation. Similar assay systems have been
utilized previously(3) . This assay also allows us to determine
if methylation affects the DNA end-joining. The assay is sensitive and
quantitative, since a large sample pool of plasmid DNA can be tested by
its ability to confer antibiotic resistance to bacteria. Even more,
individual recombination and end-joining products can be analyzed by
recovering plasmid DNA from bacterial colonies, thus providing insights
into repair processes at the molecular level.
) to bacteria (Fig. 1). The Tn5 neo gene expresses only in bacteria, so as to bypass any potential
transcriptional effects on recombination and end-joining in mammalian
cells. This plasmid and its derivatives do not contain a mammalian
replication origin. The two intermolecular recombination substrates,
M5neo and M3neo, contain 3`- and 5`-truncated Tn5 neo genes,
respectively, and have an overlap of 352 bp of homologous sequences
within the neo gene. Recombination within this homology
restores a functional neo gene. To stimulate recombination,
the plasmids are cleaved at the end of the homology region prior to
transfection. M5neo is cleaved with SphI (M5neo/S), and M3neo
is cleaved with PstI and AatII (M3neo/AP).
Restriction with each of these enzymes produces 4-base 3` overhangs,
although the sequence of the overhang differs for each.
)The recovered DNA is electroporated into E. coli, and colonies are selected on both ampicillin (Amp)
and kanamycin (Kan) plates. The design is such that kanamycin selection
should allow us to measure recombination, whereas ampicillin selection
should allow us to measure end-joining.
Preparation of DNA for Transfection into Mammalian
Cells
M5neo/S and M3neo/AP are methylated in vitro with
the cytosine methylase, M. SssI. Although of bacterial origin,
the SssI methylase mimics the mammalian methyltransferase,
methylating all cytosine residues within the dinucleotide
CpG(22) . Methylation is monitored by resistance of plasmid DNA
to digestion by HpaII, which has a CCGG recognition sequence (Fig. 3). Methylated substrates are not cleaved by HpaII, whereas unmethylated substrates are cleaved to smaller
fragments. Methylation appears to be highly efficient by this criteria.
pG, DNA methylated
with methylase SssI. AP, AatII/PstI; S, SphI; H, HindIII.
10
colonies on either Amp or Kan plates, for an
overall transformation efficiency of approximately 4 10
colonies/µg (Table 1). The efficiency was the same
whether or not the DNA was methylated.
transformants. This
is a result of powerful exonucleolytic activities present in bacterial
cells, which degrades incoming linear DNA. The resulting transformants
are likely due to a low level of repair of the broken DNA molecules by
the bacterial cells. No Kan colonies are obtained with
M5neo/S, since the neo gene contains a 3` truncation. to an even lower level than M5neo/S, possibly due to
the close proximity of the AatII site to the start of the amp gene. Thus, M3neo/AP plasmids which have recircularized
may have defective amp genes. Alternatively, differences in
the overhangs produced by restriction digestion may contribute to the
lower transformation efficiency of M3neo/AP. As with M5neo/S, M3neo/AP
does not give rise to Kan colonies due to truncation of the neo gene. colonies were
obtained at approximately the same level as that seen for M5neo/S
alone, no Kan colonies were obtained, indicating that
recombination between the two substrates is very inefficient during
bacterial transformation. These results indicate that the bacterial
transformation assay will be highly sensitive for the detection of
recombination products and that cytosine methylation will not affect
the outcome of the assay in bacteria.Transfection of Methylated DNA into COS1
Cells
COS1 cells were transfected via electroporation with the
methylated and unmethylated recombination substrates M5neo/S and
M3neo/AP and with the positive control Mneo. After an incubation of 4
h, extrachromosomal DNA was recovered from the cells and subjected to
Southern analysis. This allowed us to monitor the recovery of DNA and
the retention of methylation. A similar amount of DNA was recovered
whether or not the DNA was methylated (Fig. 4). DNA degradation
appeared to be minimal during the 4-h incubation.
pG),
transfected into COS1 cells, and recovered after 4-h incubation. A
portion of the DNA was subjected to HpaII digestion, as
indicated. The Mneo plasmid was used as the
probe.
Effects of CpG Methylation on Extrachromosomal Homologous
Recombination and DNA End-joining
To determine if methylation
affects either extrachromosomal recombination or DNA end-joining, the
COS1-transfected DNA was electroporated into E. coli DH12S (Table 2). Transfection of the parental Mneo plasmid, either
methylated or unmethylated, into COS1 cells, did not affect its ability
to confer Amp or Kan to bacteria.
Cotransfection of M5neo/S and M3neo/AP into COS1 cells, in contrast to
cotransformation into bacteria, resulted in a large number of both
Amp and Kan colonies. Approximately 10
Amp colonies and 10
Kan colonies were obtained, a 1% ratio of Kan colonies to
Amp colonies. Methylation of the DNA prior to transfection
did not affect the number of Amp or Kan colonies. Normalizing to Mneo, the yield of Amp colonies from the M5neo/S and M3neo/AP cotransfection in COS1
cells was 73-fold higher than direct E. coli transformation,
whereas the yield of Kan colonies was increased more than
2000-fold.
colonies were obtained from
transfecting M5neo/S or M3neo/AP separately into COS1 cells. In
addition, premixing the M5neo/S and M3neo/AP which had been separately
transfected upon electroporation into bacteria did not result in any
Kan colonies, such that Kan was dependent upon
cotransfection of both of the DNAs into COS1 cells. These results
demonstrate that Kan is a result of homologous
recombination between M5neo and M3neo in COS1 cells and, therefore,
that extrachromosomal recombination is not affected by CpG methylation. colonies provides a measure of DNA
end-joining in COS1 cells. Transfection of unmethylated and methylated
M5neo/S results in 1.7 10
and 2 10 colonies, respectively. Transfection of unmethylated and
methylated M3neo/AP results in 5.1 10 and 4.3
10 colonies, respectively. These are roughly 2%
(M5neo/S) and 6% (M3neo/AP) the number of colonies obtained with Mneo.
These figures are substantially higher than what was seen with direct
bacterial transformation of the plasmids (Table 1). For M5neo it
is approximately 14-fold higher, whereas for M3neo it is more than
7000-fold higher. These results indicate that the COS1 cells are able
to rejoin the broken DNA ends more efficiently than the bacterial cells
and that this end-joining is unaffected by CpG methylation of the
plasmid DNA. colonies. These results indicate that the joining of
ends differs mechanistically between mammalian cells and E.
coli. colonies
derived from COS1 cotransfection of M5neo/S and M3neo/AP for both the
methylated and unmethylated samples (Fig. 5). Similar results
were obtained. Most plasmids were identical to the positive Mneo
control (R), indicating that recombination had occurred within
both the neo gene and plasmid backbone sequences. A small
number of plasmids (RE), 1 or 2 out of 30, had undergone
recombination only within the neo gene. DNA end-joining had
occurred to join the molecules within the plasmid backbone.
or Kan
(Amp)
colonies and its structure determined by restriction enzyme
digestion.
/Amp colonies from the cotransfection. One
class of end-joining products (5E) had precisely rejoined the
cohesive SphI ends of M5neo/S. This class consisted of three
plasmids derived from the unmethylated sample and one plasmid from the
methylated sample. The more numerous class of end-joining products (3E2) was derived from DNA end-joining of the AatII/PstI plasmid backbone fragment of M3neo/AP.
This class consisted of seven plasmids from the unmethylated sample and
nine plasmids from the methylated sample. Based on restriction
analysis, most of the plasmids in this class contained only a limited
modification of the AatII/PstI ends. AatII
has a 3` ACGT overhang and PstI has a 3` TGCA overhang. The
rejoining of these two heterologous ends is not unexpected, given the
promiscuity of end-joining in mammalian cells (2) .Direct Examination of Recombination and End-joining
Products
Bacterial transformation is sensitive yet indirect for
assaying recombination and end-joining processes in mammalian cells. To
directly monitor recombination and end-joining in COS1 cells, recovered
plasmid DNA was analyzed by Southern blot analysis. A variety of
products are expected in addition to those already observed by plasmid
recovery (Fig. 6A). Recombination between M5neo/S and
M3neo/AP can occur within either the neo gene homology (RE) or the plasmid backbone sequences (ER) or both (R). Similarly, end-joining can occur at the ends of the neo gene homology (ER), the plasmid backbone (RE), or both (EE). In addition, intramolecular
end-joining products are also possible. The two M3neo/AP fragments can
recircularize to form 3E1 and 3E2, although the latter is not detected
since it does not hybridize to the neo probe. M5neo/S can
recircularize to form 5E. (Although not illustrated, similar junctions
can also be formed in these three cases by intermolecular end-joining
of two identical fragments.) One other intermolecular junction is
shown. This is formed by end-joining the two neo fragments
from M5neo/S and M3neo/AP in opposite orientations (E).
)As with mammalian cells,
transfection studies of plant protoplasts have demonstrated that CpG
methylation does not affect extrachromosomal
recombination(23) .
However, considering the
nonconservative nature of most recombination events between transfected
DNAs(31) , these results are inconclusive regarding the double
strand break repair pathway.
)))
We thank members of the Jasin laboratory, especially
Rich Bennett, Peter Romanienko, Philippe Rouet, and Fatima Smih, for
assistance in various facets of this project.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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