Received 21 July 1999/Returned for modification 10 September
1999/Accepted 27 September 1999
A double-strand break (DSB) in the mammalian genome has been shown
to be a very potent signal for the cell to activate repair processes.
Two different types of repair have been identified in mammalian cells.
Broken ends can be rejoined with or without loss or addition of DNA or,
alternatively, a homologous template can be used to repair the break.
For most genomic sequences the latter event would involve allelic
sequences present on the sister chromatid or homologous chromosome.
However, since more than 30% of our genome consists of repetitive
sequences, these would have the option of using nonallelic sequences
for homologous repair. This could have an impact on the evolution of
these sequences and of the genome itself. We have designed an assay to
look at the repair of DSBs in LINE-1 (L1) elements which number
105 copies distributed throughout the genome of all
mammals. We introduced into the genome of mouse epithelial cells an L1
element with an I-SceI endonuclease site. We induced DSBs
at the I-SceI site and determined their mechanism of
repair. We found that in over 95% of cases, the DSBs were repaired by
an end-joining process. However, in almost 1% of cases, we found
strong evidence for repair involving gene conversion with various
endogenous L1 elements, with some being used preferentially. In
particular, the TF family and the L1Md-A2 subfamily, which
are the most active in retrotransposition, appeared to be contributing
the most in this process. The degree of homology did not seem to be a
determining factor in the selection of the endogenous elements used for
repair but may be based instead on accessibility. Considering their
abundance and dispersion, gene conversion between repetitive elements
may be occurring frequently enough to be playing a role in their evolution.
 |
INTRODUCTION |
DNA double-strand breaks (DSBs)
represent a threat to genomic integrity that have to be repaired
efficiently or they will lead to serious consequences, such as
programmed cell death (45). In mammalian cells, a single DSB
can cause a cell cycle arrest (17). Mammalian DSB repair is
a complex process that involves multiple proteins that vary depending
on the cell cycle phase (16). Two main repair pathways are
utilized (22). The first one involves the rejoining of the
broken ends with or without the loss and/or addition of DNA. The second
one involves homologous recombination with an intact copy of the broken
DNA segment acting as a template. In most cases the homologous template
could be the allelic copy present on the sister chromatid or homologous chromosome. However, since 30% of the genome is made of highly repetitive sequences, this raises the possibility that nonallelic homologous templates could be used for the repair of a DSB. Indeed, several groups have shown that a DSB occurring in a chromosomal construct containing tandemly repeated sequences could be repaired by
homologous recombination at a frequency of as high as 30% (7, 12,
21, 22, 39, 44). Also, it was shown that homologous sequences
located at a nonallelic position on a distinct chromosome could be used
for DSB repair, albeit at a much lower frequency (34).
We wanted to determine how a DSB in highly repetitive sequences would
be repaired. In particular, we were interested in the repair of a DSB
occurring in LINE-1 (L1) elements. L1 elements are present in all
mammals at around 105 copies dispersed throughout the
genome and are more conserved within a given species than between
closely related species (18). This concerted evolution of L1
elements could be due in part to homologous recombination between
nonallelic L1 elements. Indeed, Saxton and Martin (40)
suggested that recombination between particular L1 subtypes in mouse
could have created the TF family. There is some evidence
that neighboring L1 elements can be involved in homologous
recombination leading to chromosomal deletions (9, 43).
Also, recombination between L1 elements would be partly responsible for
the present hominid Y chromosome structure (42). However, it
could not be determined if DSB repair was implicated in these processes.
In order to study DSB repair of L1 elements, we devised an assay based
on the I-SceI system (19, 36). We integrated at several distinct sites in the genome of a mouse epithelial cell line an
L1 element containing an I-SceI site and then introduced a
DSB at that site by the transfection in these cells of a vector expressing the I-SceI endonuclease. The results were that,
over 95% of the time, the DSBs in the L1 sequences were repaired by an
end-joining process. However, in almost 1% of the cases we found
strong evidence for homologous recombination repair involving gene
conversion with endogenous L1 elements. Several distinct endogenous L1
elements were used for homologous recombination repair, some
preferentially. In particular, the TF family and L1Md-A2
subfamily, which are currently the most active in retrotransposition, appeared to be contributing most of the elements in these gene conversion events.
| |
MATERIALS AND METHODS |
Cells and growth conditions.
Nod-2 are nonciliated
epithelial cells of the bronchioles derived from a pulmonary
adenocarcinoma (nodule) of an FVB transgenic mouse that harbors a
polyomavirus large-T-antigen (T-Ag) transgene (20). Nod-2
cells are probably derived from precursor cells within the basal
bronchiolar epithelium. Cells were maintained in Dulbecco modified
Eagle medium (DMEM) medium supplemented with 10% fetal bovine serum
plus 50 µg of gentamicin per ml and kept in a 5% CO2
atmosphere at 37°C. When used, hygromycin was added at a final
concentration of 150 µg/ml.
Vector construction.
pASB-SceI is derived from
pASB-HindIII which contains the 3-kb
KpnI-EaeI fragment of the BALB/c L1Md-A2
(4). An I-SceI restriction site was introduced at
the unique HindIII site by using an oligodimer (see Fig.
1b).
Transfection.
A total of 2 × 106 nod-2
cells were transfected in DMEM by electroporation with 10 µg of
NarI-digested pASB-SceI and 1 µg of BamHI-linearized hygromycin vector, p3'SS (Stratagene). The
electroporation conditions used were as follows: 0.4-cm gap chamber,
960 µF, and 270 V. Hygromycin was added 48 h after the
electroporation, and Hygr colonies were picked 3 weeks later.
Transient I-SceI expression.
pCBASce
(34) is an expression vector that contains the coding
sequences of the I-SceI endonuclease from
Saccharomyces cerevisiae, under the control of the 
-actin
gene promoter. A total of 4 × 106 cells in DMEM of
the different pASB-SceI containing clones were electroporated (see conditions in the previous section) with 100 µg
of uncut pCBASce vector or without vector as a control. Viable cells
were harvested 72 h later for genomic DNA extraction and analysis.
DNA analysis.
Southern analysis was performed by using 13 µg of digested genomic DNA according to Ausubel et al.
(3). The NarI-KpnI polyomavirus origin
fragment of pASB-SceI was used as a probe. All of the
restriction enzymes used were purchased at New England Biolabs except
Meganuclease I-SceI (Boehringer Mannheim). For all PCR
amplifications, the Expand High-Fidelity PCR System (Boehringer
Mannheim) was used. The primers used for PCR were as follows: 1, 5'-CAGAGGAGGTGTATGGGTTTGTC-3'; 2, 5'-CGAGTCAGTGAGCGAGGAAGC-3'; 3, 5'-GTTTGTAAGTCGAACAGCGGGGGCTATATG-3'; and 4, 5'-TCTCCCCGCGCGTTGGCCGATTCATTAATG-3'. PCR1 (primers 1 and 2)
amplification was performed on 150 ng of digested genomic DNA as
follows: 94°C for 2 min; 10 cycles of 94°C for 1 min, 62°C for 1 min, and 68°C for 2 min; and 20 cycles of 94°C for 1 min, 62°C
for 1 min, and 68°C for 2 min (plus a 20-s/cycle). For PCR2 (primers
3 and 4), the same procedure was followed except that the annealing
temperature was changed from 62 to 70°C. For DNA sequencing of PCR
products, PCR fragments were cloned (KpnI-SacI) or subcloned (KpnI-HindIII) into pBluescript
SK(+) (Stratagene). Sequencing was done by the dideoxy chain
termination reaction (38) by using Sequenase (Amersham).
Sequence data were compiled and analyzed by using Genetic Computer
Group software.
| |
RESULTS |
Experimental design.
The strategy that we used was to
introduce in the genome of a mouse cell line, an L1 fragment in which
we had replaced a highly conserved restriction site by an
I-SceI site. We then created a DSB at the I-SceI
site in vivo and scored for repair events by the loss of the
I-SceI. To enrich for homologous recombination repair
events, we selected for the reacquisition of the highly conserved
restriction site.
The vector we used has been described previously (4). It
contains a 3-kb fragment from the 3' end of the well-characterized L1Md-A2 element (Fig. 1a). We chose the
3'-end fragment since most L1 elements of the mouse genome are
truncated at their 5' end (47). Certain restriction sites
are highly conserved in mouse L1 elements, and this is the case for the
HindIII site present in the L1 fragment we used, which
is present in more than 50% of the mouse L1 elements (4,
8) (GenBank-EMBL L1 sequences). We introduced in the
HindIII site an I-SceI cassette to create the
vector pASB-SceI (Fig. 1b).
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FIG. 1.
Structure of L1Md-A2 (23) (a) and the
pASB-SceI vector used in our assay (b), which contains the
3-kb KpnI-EaeI fragment of L1Md-A2. A linker
containing the I-SceI restriction site was introduced at the
HindIII site: H' designates the partial
HindIII sites flanking the I-SceI cassette,
and the I-SceI cut site is shown. oriPy, polyomavirus origin
that was present in the initial pASB-HindIII vector
(4). Position of PCR primers (1 to 4) and sequencing primers
(5 to 8) in pASB-SceI are as follows: 1 (5850 to 5872),
2 (3090 to 3070), 3 (5887 to 5916), 4 (3043 to 3014), 5 (1500 to 1481),
6 (1107 to 1087), 7 (872 to 850), and 8 (562 to 542).
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pASB-SceI was transfected in a mouse epithelial cell line
(nod-2 [20]), along with the hygromycin selection
vector p3'SS. Three independent clones were selected (clones 19, 22, and 25); each had integrated into their genome a single complete
copy of the NarI-digested pASB-SceI vector,
as indicated by the presence of a single complete insert seen by
Southern analysis (Fig. 2). We then
created a DSB in the integrated L1 sequence in clones 19, 22, and 25 by
transfecting an I-SceI expression vector (pCBASce [34]).
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FIG. 2.
Southern blot analysis of the PstI-digested
genomic DNA isolated from clone 19 (lane 3), clone 22 (lane 4), and
clone 25 (lane 5) derived from nod-2 cells transfected with
pASB-SceI and p3'SS, and from parental nod-2 cells (lane 1)
and nod-2 transfected with the selection vector p3'SS alone (lane 2).
The NarI-KpnI polyomavirus origin fragment of
pASB-SceI was used as a probe (Fig. 1b).
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To analyze the repair events, the genomic DNA was first digested with
PstI to break down the genome and I-SceI to
cleave the integrated L1 sequences which had not lost the
I-SceI site. Integrated L1 sequences which had lost the
I-SceI site were then amplified by PCR with primers 1 and 2 (Fig. 1b). The resulting PCR products were digested with
KpnI and SacI, cloned, and sequenced by using primer 5. To enrich for homologous recombination events involving the
reacquisition of the HindIII site, after the first PCR
the procedure was repeated (I-SceI digestion and PCR
amplification) with internal primers 3 and 4. The resulting products
were digested with KpnI-HindIII, cloned, and
sequenced from the HindIII site, with primers flanking
the cloning site and primers 6, 7, and 8 if necessary.
Nature of the repair events.
The I-SceI cleavage
efficiency was determined for each of the six independent pCBASce
transfection assays performed and was found to vary depending on the
clone used. After each transfection with the pCBASce vector, the
genomic DNA was extracted and digested with PstI, amplified
by PCR with primers 1 and 2 (Fig. 1b), digested with I-SceI,
and analyzed on a gel. The relative proportion of digested to
nondigested DNA was quantified by densitometry. For clones 19 (three
assays) and 25 (one assay), ca. 35% of the PCR products were resistant
to I-SceI digestion, while for clone 22 (two assays), only
ca. 20% were resistant to I-SceI digestion (Fig.
3). Thus, for clones 19 and 25, 35% of
the integrated L1 sequences were involved in a repair event, while it
appears that for clone 22 only 20% were involved in a repair event. It
should be noted that it is not possible with this assay to determine if
repair events occurred that would have regenerated the
I-SceI site.
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FIG. 3.
I-SceI digestion of PCR1 products amplified
from PstI-digested genomic DNA isolated from the
pCBASce-transfected clones 19 (lane 1), 22 (lane 2), and 25 (lane 3).
Lanes c correspond to the same clones subjected to electroporation but
in the absence of pCBASce. This control indicates that the
genomic DNA from each clone could be digested by
I-SceI. The I-SceI digestion of the PCR1 product
of 3 kb should yield bands of 1.7 and 1.3 kb. In clones 19 (lane 1) and
25 (lane 3), ca. 35% of the PCR product is resistant to
I-SceI digestion. In the case of clone 22 (lane 2), only
20% of the PCR product is resistant to I-SceI digestion.
The numbers at the side of the figure indicate the molecular weight.
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The largest number of repair events were analyzed from clone 19. Of 111 repair events defined by the loss of the I-SceI site, 4 had
deletions larger than 1 kb and 81 had small deletions (on average, 10 bp), including 6 with additions of small DNA fragments. Another 20 repair events had additions of small DNA fragments without deletion.
Thus, at least 105 of the 111 repair events (95%) were the result of
end joining. The remaining six repair events involved the acquisition
of an HindIII site replacing the I-SceI
inserted sequences. To determine if this was reproducible, PCR products
from other assays with clones 19, 22, and 25 were tested for the
acquisition of the HindIII site. As in the initial experiment, HindIII-acquiring events were also found
to represent fewer than 5% of the events in each case, as evaluated by
restriction enzyme analysis. The HindIII-acquiring
events were due to DSB repair, since we were not able to isolate
HindIII-acquiring events in controls not transfected
with pCBASce.
To enrich for homologous recombination events, 304 HindIII-acquiring events were cloned and sequenced
from six independent pCBASce electroporation assays. From these, 20 had
acquired an HindIII site as a result of a fortuitous
deletion of vector sequences around the I-SceI site that
generated a de novo HindIII site or as a result of the
acquisition at the I-SceI site of a pCBASce fragment that
contained an HindIII site. Of the remaining 284 HindIII-acquiring events, the vast majority (252)
had sequences identical to that of the L1 sequences present in our
vector (or with 1 or 2 random base changes per 300 bp; see below).
Since, on average, endogenous mouse L1 elements differ in their
sequences from one another by 4% (24) and since a number of
polymorphic bases are located near the HindIII site
(Table 1), a gene conversion event might
be expected to generally introduce other base changes beside the
acquisition of the HindIII site. Instead, these were most probably generated by end-to-end joining at the two half HindIII sites bordering the I-SceI cassette
(see H'; figure 1b) rather than gene conversion events. In this case,
end-joining would occur at the 4-bp H' repeat, perhaps by a
single-strand annealing type of mechanism that uses such
microhomologies. Indeed, in our analysis of the 111 PCR products
described above, we found single-strand annealing-type events at
repeats of 2 to 4 bases at about the same frequency (8 of 111) as the
HindIII-acquiring events (6 of 111). Previous
studies had already shown that repair events involving strand annealing
of a few bases are a common end-joining repair process (11,
28, 29, 31, 35), including those occurring at short repeats
flanking an I-SceI site (37).
The remaining 32 HindIII-acquiring events appeared
to be bona fide gene conversion events with endogenous L1 elements,
based on at least two of the following criteria: (i) they had at least one base change beside the acquisition of the HindIII
site; (ii) specific base changes were repeated in different
HindIII-acquiring events originating from different
PCR amplifications; (iii) the base changes could be regrouped in
conversion tracts of different lengths; (iv) the base changes were
skewered toward the HindIII site; (v) the base changes
corresponded to diagnostic bases of various L1 subfamilies.
Since base changes could be produced as an artifact of PCR, we did a
control experiment to evaluate the number of base changes that could be
attributed to PCR. We spiked nod-2 genomic DNA with 1 copy per
genome of the pASB-SceI plasmid and followed the
experimental procedure described above. Of 5,225 bp sequenced from 20 different amplified pASB-SceI molecules, we found altogether
seven base changes located at random positions. Thus, we excluded from
the conversion events infrequent HindIII-acquiring
events that had only one or two base changes that did not repeat
themselves in an independent PCR amplification.
Taken together, these results indicate that 5% (6 of 111) of the
repair events were HindIII-acquiring events. From
these, 11% (32 of 284) could be identified as bona fide homologous
recombination repair events, giving an overall homologous recombination
frequency of almost 0.6%. It is possible that we are underestimating
the frequency of gene conversion events, since those occurring with endogenous L1 elements without an HindIII site would not
be part of the HindIII-acquiring events. However,
these are apparently infrequent, since none were seen in the 111 events
analyzed. Also, events with extremely short conversion tracts, i.e.,
those that did not extend past the HindIII site to the
first mismatched base between the endogenous L1 element and the
pASB-SceI L1 sequence, would not have been scored as a
conversion event. Short conversion tracts have been observed in other
studies involving the repair of chromosomal DSBs (13, 44).
Nevertheless, the proximity and abundance of polymorphic bases near the
HindIII site should have permitted the detection of most
events (Table 2).
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TABLE 2.
Categorization of the 32 gene conversion events with
respect to the predominant L1 polymorphic bases from the
HindIII sitea
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Gene conversion events with endogenous L1 elements.
The
conversion tracts of the 32 gene conversion events that were identified
in this study are presented in Table 1. The extent of conversion from
the HindIII site that could be scored by the presence of
base changes was as short as 13 bp or as long as 780 bp, with the
majority being shorter than 100 bp. The conversion tracts occurred
between L1 sequences that were as much as 10% heterologous, with most
being less than 4%.
For each clone, several different patterns of gene conversion were seen
that involved distinct endogenous L1 partners (11 of 21 for clone 19, 5 of 5 for clone 22, and 5 of 6 for clone 25). In at least one instance,
it appeared that several independent gene conversion events involved
the same endogenous L1 partner, resulting in a set of nested conversion
tracts (the first 10 gene conversion events for clone 19).
Interestingly, the longest conversion tract of this cohort (783 bp,
event 19-0.01), which has incorporated 24 base changes compared to
pASB-SceI, is 100% identical to the sequence of a L1
element that has recently been reported as actively being involved in
retrotransposition events (L1spa, AF016099 GenBank/EMBL
accession number) (27).
Because their sequences are identical and they come from the same
pCBASce electroporation assay (but not the same PCR amplification), gene conversion events 19-1.02, 19-2.08, 19.4.06, and 19-8.01 could
represent the same clonal event, as could be the case for events
19-E.05 and 19-B.07 or events 25-1.07 and 25-5.03.
Comparative analysis of mouse L1 elements indicate that they can be
divided into at least three groups or families of more-related sequences that have been designated the F, A, and TF
families (10). The F family would be the oldest and most
diverged one, while the A and TF families would be more
recent, with a higher degree of homology among their members (97 and
99.8%, respectively). The TF family would possibly be the
most active in retrotransposition (27). The L1 sequences
present in pASB-SceI are derived from L1Md-A2
(23), which is part of an A subfamily in which members share
99.5% of homology and are also active in retrotransposition (41). The TF family and the L1Md-A2 subfamily
have been estimated to contain the same number of full-length members,
i.e., 2,000 to 3,000 per diploid genome (40). The L1
segments that were sequenced in this study include a number of
diagnostic bases that are identified with the L1Md-A2 subfamily or
TF family. These are indicated in Table 2. Analysis of the
gene conversion tracts we observed indicated that the majority of
events can be classified as possibly having involved a L1Md-A2
subfamily partner (17 events) or a TF partner (10 events).
Five events have a mixed TF and A subfamily conversion
tract that could have arisen from either mismatch repair or
recombination with a mosaic TF/A L1 partner (40).
Gene conversion event 22-H.03 has an interesting structure (Fig.
4a) that suggests a one-sided invasion
homologous recombination event. This event has an 118-bp L1 duplication
containing 9-bp changes predominantly of the TF pattern,
and a deletion of 12 bases from the I-SceI cassette. Figure
4b illustrates the different steps that have possibly been involved in
the genesis of event 22-H.03, based on the one-sided homologous
recombination model (4, 5). The steps are invasion of the L1
donor sequence by only one side of the double-strand break, followed by
DNA synthesis and then release of the invading end, and end-to-end
joining with the other side of the DSB. This had also been observed in
earlier studies on homologous recombination between extrachromosomal
and endogenous chromosomal L1 elements in mouse cells (4,
6).
| |
DISCUSSION |
In the present study, we have shown that a DSB in LINE elements
could be repaired by homologous recombination with nonallelic endogenous L1 elements. To our knowledge, this is the first
demonstration of homologous repair of a chromosomal DSB by endogenous
genomic repeat sequences in mammal cells. However, repair of a
DSB in Ty elements by nonallelic Ty chromosomal sequences has been
reported in yeast cells (30). We found that the dominant
repair mechanism leading to I-SceI site loss was by
far end joining rather than gene conversion with an endogenous L1
element. This contrasts with yeast, in which nonallelic repetitive
elements are readily used (30). Nevertheless, considering
the large number of repetitive elements, a frequency of almost 1% DSB
repair by homologous recombination with nonallelic L1 elements
could be enough to have played a role in the evolution of repetitive
elements. In this regard, it is interesting that the selection of
partners was not based primarily on the degree of homology, since
partners with higher (L1Md-A2 subfamily) and lower (TF
family) homology appeared to have participated equally in the process.
A lack of preference for highly homologous partners had also been seen
for homologous recombination between extrachromosomal and chromosomal
L1 elements (6, 33). This could explain the genesis of
mosaic lineages in L1 elements (40).
The choice of partners for DSB repair by homologous recombination could
be more a question of accessibility than homology. We do not know the
relative location of the endogenous L1 partners relative to our
pASB-SceI L1 element. It could have been intrachromosomal proximal or distal, or interchromosomal, or even extrachromosomal. This
last possibility seems less likely since what appears to be the same
partner was in some cases involved repeatedly in the process, resulting
in a set of encompassing conversion tracts (see clone 19, Table 1). In
any case, several partners were clearly accessible for DSB repair.
It would seem that the most likely endogenous L1 partners were members
of the L1-MdA2 subfamily or the TF family. These represent only a fraction of the total L1 elements of the mouse genome. The
full-length members in these two groups have been estimated at 2,000 to
3,000 for each (40). However, they are by far the most
active in retrotransposition (10, 27, 40, 41). It is
tempting to speculate that this could somehow influence their accessibility. In particular, we observed for one endogenous L1 partner a 100% homology over 800 bp with a TF element,
L1spa, that has been shown to be currently involved in
retrotransposition (27).
In one case, we observed that the DSB repair involved a gene conversion
event with a structure compatible with a one-sided invasion (OSI)
mechanism, as we had described previously (4). OSI appears
to be a universal homologous recombination mechanism seen in mammals
(1, 4, 14, 26, 34), yeasts (15), plants
(32), insects (46), and birds (25). It
can lead to the transfer of several kilobases of nonhomologous
sequences between nonallelic homologous sites (2, 4,
15). Thus, OSI between L1 elements could have had an impact
on the evolution of the mammalian genome, permitting the transfer of
unique sequences from one location to another via repetitive elements
that would provide numerous entry points. In this regard, it is
interesting to note that we have readily detected homologous
recombination between nonallelic L1 elements in germ cells of
transgenic mice (A. Tremblay et al., unpublished data).
We gratefully acknowledge Dominic Corbeil, Hélène
D'anjou, Julie Guay, Nadine Gusew, Benoit Houle, Roxane Lussier, and
Hugo Würtele for their technical assistance. We also thank
Anne-Marie Mes-Masson for her interest in this work and for providing
the nod-2 cells.
This study has been supported by a grant from the Medical Research
Council (MRC) of Canada. A.T. is a recipient of an MRC fellowship.
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Abstract
Introduction
