(Received for publication, July 18, 1995; and in revised form, December 18, 1995)
From the
The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40-transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co-transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co-transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra-chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA.
Ataxia telangiectasia (A-T) ()is an autosomal
recessive disorder characterized by progressive cerebellar ataxia, skin
changes, endocrine disorders, immune defects, and high risk of cancer.
A-T cell lines have abnormalities in DNA damage processing including
hypersensitivity to ionizing radiation (1) and
radiation-resistant DNA synthesis(2, 3) . The A-T gene
function appears to be central to DNA damage processing, cell cycle
checkpoints, and genomic stability; interest in determining the
function of the gene comes from many disciplines.
Following localization of the A-T gene to 850 kb on the long arm of chromosome 11(4, 5) , it now appears to have been cloned(6) . The surprising result is that one gene is mutated in all the complementation groups, which makes the original observations of complementation groups difficult to rationalize. The cloned sequences have features in common with a phosphatidylinositol 3-kinase and the rad 3 gene of Schizosaccharomyces pombe. How loss of function of this gene achieves the pleiotropic phenotype remains unsolved.
Progress in understanding this complex syndrome has been restricted to extensive definition of the phenotype(7) . One of the main puzzling aspects of the A-T response to ionizing radiation is the lack of a gross defect in DNA double-strand break repair. Recently it has been suggested that all or most of the ionizing radiation sensitivity of A-T is due to early triggering of programmed cell death by otherwise non-lethal DNA damage(8) . Another view of the A-T gene function is a lack of damage surveillance, which may help explain both radiosensitivity and lack of cell-cycle arrest in response to ionizing radiation. The role of the A-T gene in the damage signaling pathway was suggested by the lack of or delayed rise in p53 following ionizing radiation(9, 10, 11, 12) . However, the models proposed by both Meyn (8) and Thacker (7) are incongruous: if the damage signaling pathway is at fault, how is apoptosis triggered so readily? Signaling of damage appears sufficient to stimulate known response markers such as poly(A)DPribose(13) .
Although A-T cells have no gross defect in closing double-strand breaks, they have been shown to have a high frequency of double-strand break mis-repair (14, 15, 16) using plasmid reactivation. A high rate of spontaneous intra-chromosomal, but not extra-chromosomal, recombination has also been described using a plasmid recombination substrate(17) . Our previous observations had suggested that mis-repair may be linked to recombination, and a working hypothesis was that double-strand breaks from ionizing radiation trigger recombination, which in A-T is error prone. Thus, although strand breaks are closed readily, residual damage is present which leads to mitotic cell death or apoptosis. The cell-cycle arrest response to ionizing radiation may be more complex than first described, and was recently shown to be cell-cycle phase dependent(18) . The A-T gene may highlight a difference in the signal pathway of cell-cycle arrest and apoptosis.
In this report we transfected plasmids, designed to evaluate DNA recombination/repair events, into A-T and normal cell lines. Specifically, we measured chromosomal and extra-chromosomal recombination; whether cleavage of the recombination substrates affected the outcome; and the type of recombination events seen in A-T compared with normal cells.
Figure 1:
A, map of plasmid pTPSN linearized at
the ClaI site showing relevant restriction enzyme sites. The
linear plasmid is 10.2 kb. Two mutant hygromycin genes (hyg)
are enclosed within different sized DraI fragments: a HindIII insertion at SacII site cuts the 2.6-kb DraI fragment into 1.4- and 1.2-kb bands; a HindIII
insertion at PvuI site cuts the 2.9-kb fragment into 1.7- and
1.2-kb bands. There are two HindIII fragments: a 4-kb HindIII fragment containing the single ClaI site and
the remaining 6-kb HindIII fragment contains the neomycin
resistance gene (neo). The shaded areas represent the
gene promoter regions. The solid black line represents the HindIII linker insert. The arrows show the direction
of transcription. B, BamHI; C, ClaI; D, DraI; H, HindIII.
Relevant restriction fragment lengths in kb are shown. B, map
of plasmid pSV2gpt showing the deletions within the
xanthine-guanine-phosphoribosyl transferase gene (xgprt) which
give rise to the p2 and p
3 plasmids. p
2 has the PvuII-HindIII fragment deleted; p
3 has the EcoRV-BglI fragment deleted: the deleted parts of the
gene are shaded. Between the sites of deletion in p
2 and
p
3, there are 460 base pairs of identical sequence, where
recombination will lead to a functional xgprt gene. The KpnI and EcoRV restriction enzyme sites are within
the xgprt gene and the EcoRI restriction enzyme site
is distant from the xgprt gene by
2 kb in either
direction.
The
plasmids p2,p
3 (20) were derived from
pSV2gpt(21) and are shown in Fig. 1B.
These plasmids contain the bacterial amp gene and the gpt gene with a deletion. The bacterial gpt gene with
flanking SV40 promoter and processing sequences, confers resistance to
mycophenolic acid. p
2 lacks the SV40 promoter and the first 120
base pairs of the gene coding sequence, whereas p
3 lacks the 3`
end of the gpt gene. There are 460 base pairs between the
deleted sequences, retained in both deletion plasmids, containing the
single KpnI site. A single EcoRI restriction enzyme
site is distant from the gpt coding region. The deletion
plasmids do not have an intact gpt gene, but if
p
2+p
3 are co-transfected into cells, reconstitution of
the gpt gene can occur via inter-molecular recombination.
Single G418-resistant (neo) colonies were
isolated and amplified with and without G418. In parallel, the clones
were tested for hygromycin sensitivity and only hygromycin-sensitive
clones were used for further testing. The initial cell count at the
time of establishing hygromycin sensitivity and the final cell count
following amplification, prior to seeding in hygromycin, were used to
calculate the recombination frequency per cell generation over which
recombination events could accumulate(22) , taking no account
of cell loss following cell division, but correcting for plating
efficiency. Mixed G418-resistant (neo
) colonies were also
harvested and counted: if the cell number was insufficient for
immediate seeding, amplification with or without G418 was allowed. The
recombination frequency was corrected for cell generation based on the
total number of G418-resistant colonies harvested and the calculated
amplification of cells prior to seeding in hygromycin. When there were
sufficient cells amplified from either single or multiple
G418-resistant colonies, 10
, 10
, and 10
cells were seeded into T75 flasks in medium with or without G418,
and 50 µg/ml hygromycin was added the next day.
After
14-21 days growth in hygromycin, visible colonies
(hyg) were stained with methylene blue. The frequency of
hygromycin resistance per viable cell was recorded. A minimum of three
flasks per seeding density and two different seeding densities were
used per data point. Single neo
and hyg
colonies were isolated and transferred from dishes to small wells
and maintained with the same selective medium until DNA extraction.
Transfection frequencies for pTPSN and recombination
frequencies for mixed colonies are shown in Table 2. The
transfection frequencies (neo colonies per viable
cell) in AT5(BIVA), AT2(SF), and NF were not significantly different.
The geometric mean rate (± S.E.) of spontaneous chromosomal
recombination observed directly following transfection (hyg
colonies per viable neo
cell) in AT5 was 378 (245-582)
10
per cell generation, 126 times more frequent than NF, which was
2.99 (1.6-5.6)
10
per cell generation (p < 0.001). The recombination rate for AT2 was 250
(119-525)
10
per cell generation, 84
times more frequent than NF (p < 0.001). These data are not
corrected for pTPSN copy number, which did not differ significantly and
would not account for the differences between A-T and NF (see below and Table 4). Recombination frequencies were increased, 9.3 times in
AT5 and 11.8 times in NF, compared with the recombination frequency of
stable integrated sequences ( Table 1versusTable 2). At first sight, this might imply that
recombination occurring extra-chromosomally or with integration is not
significantly different between A-T and normal cells.
Figure 2: Processing of pTPSN to achieve hygromycin resistance. The right-hand lanes show plasmid only marker lanes with digestion by either HindIII or DraI or both; each pair shows 1 copy and 5 copies. When the plasmid remains unchanged, HindIII digestion results in 6- and 4-kb bands; DraI digestion leads to a 2.9- and 2.6-kb bands; and digestion by both enzymes leads to 1.7-, 1.4-, and 1.2-kb bands. Three representative clones are shown: each clone has 3 lanes reflecting HindIII, DraI, and HindIII + DraI digestion. Gene conversion leads loss of the HindIII site within either copy of the hyg gene, resulting in the 2.9-kb or the 2.6-kb band being resistant to HindIII (AT5 and AT2 clones). Reciprocal exchange results in a 2.5-kb DraI fragment which is HindIII resistant (NF clone). Other rearrangements leading to hygromycin resistance can occur and result in bands of different size with the HindIII insertion mutation removed.
Clones derived from the A-T and NF cell lines, which were resistant to hygromycin, are shown in Table 4, with a significant proportion of these clones shown in Fig. 3. The plasmid copy number in AT5 and NF was not different. AT2 clones had a single copy in 11/14 analyzed, which was lower than AT5 or NF and may explain why the recombination frequency was marginally lower in AT2 compared with AT5. However, differences in copy number cannot explain the differences in recombination frequency between A-T and NF. There was no detectable difference in the type of plasmid processing seen in clones containing a single copy of pTPSN compared with clones with multiple in tandem copies. For example, single copy status of clone C57 (AT5) was established by BamHI digest, which revealed a 2.2-kb band and one further larger band containing the other hygromycin gene (see Fig. 4A). Hygromycin sensitivity was confirmed by a failure to grow in selective media and cleavage of all DraI fragments by HindIII. Following the development of hygromycin resistance, the 2.6-kb DraI fragment has been lost, and a 3.1-kb DraI fragment developed. It is presumed that the combination of newly developed bands (3.1-kb DraI fragment; 1.9-kb DraI/HindIII fragment) accounts for hygromycin resistance, although the retention of a HindIII site in the DraI fragment is not usually found. A sequential analysis of AT5 clone C58, which contains 2 full copies of the plasmid and a further partial copy, is shown in Fig. 4B. Before selection with hygromycin, 2.9 and 2.6-kb DraI fragments are seen, which cleave with HindIII to the 1.7-, 1.4-, and 1.2-kb fragments. Additional DraI bands (5.4 kb and 3.1 kb) are seen which are both cleaved by HindIII. Following the development of hygromycin-resistance in a clone growing out of the previously cloned cell culture, the following changes are apparent: the loss of the 2.9- and 2.6-kb bands, the development of a HindIII resistance in the 3.1-kb band, and the new development of HindIII-resistant 3.7- and 1.8-kb DraI bands. Thus, in both single copy and multiple(2, 3) copy transfected clones, the development of post-integration aberrant rearrangements are a characteristic feature of A-T cells, not seen in normal cells. Hygromycin-resistant clones were obtained from multiple independent G418-resistant cultures to avoid the analyzed clones being siblings. The results showed remarkable consistency within cell type and difference between cell types; no effect of independent culture could be found.
Figure 3: A, the processing of pTPSN in 10 hygromycin-resistant clones of A-T cells (see Table 4). Two types of restriction enzyme digestion of genomic DNA were analyzed for each single cell-derived hygromycin-resistant clone: the left-hand lane of the pair shows HindIII + DraI digestion; the right lane shows DraI digestion alone. Clones C7 and W4 show gene conversion of the PvuI mutant hygromycin gene (2.9 kb, HindIII resistant). C7 shows marked amplification in addition to conversion and aberrant rearrangements. W4 also shows retention of the HindIII site in some copies of the hyg gene. C54 shows reciprocal exchange with a 2.5-kb band which is HindIII resistant, together with other bands which are abnormal but retaining the HindIII site. All other lanes show an abnormal sized DraI fragment (of sufficient length to contain a functional gene) with loss of the HindIII site. The presence of multiple faint bands may be caused by heterogeneity arising during expansion of cell number: an indicator of the instability of the genome in A-T cells. B, the processing of pTPSN in 8 hygromycin-resistant clones of NF cells. 7/8 clones show unaltered restriction-enzyme digestion patterns in one or more copies of the plasmid, together with gene conversion of one copy of the 2.9-kb DraI fragment in six of these seven clones. W5 also shows gene conversion of the 2.6-kb DraI fragment. The clone C6, which has lost all normal sized bands, showed an abnormal rearrangement with large DraI fragment (not shown).
Figure 4: A, sequential analysis of a G418-resistant clone which starts as sensitive to hygromycin and develops resistance. Single copy status is suggested by the BamHI digestion pattern (2.2-kb band and a second larger band). Both DraI fragments are cleavable by HindIII initially. As hygromycin resistance develops, a 3.1-kb DraI band appears in place of the 2.6-kb band. Curiously, the band was still HindIII cleavable, and the resultant 1.9-kb band is sufficiently large to contain the uninterrupted hygromycin sequence. B, sequential analysis of a hygromycin-sensitive clone containing 2-3 copies of pTPSN. Initial integration has generated aberrant DraI bands of 5.4 and 3.1 kb, and two normal copies of the 2.9- and 2.6-kb DraI fragments, all of which cleave with HindIII. The development of hygromycin resistance results in loss of the 2.9- and 2.6-kb DraI fragments, the development of HindIII resistance in the 3.1-kb band, the new development of aberrant sized HindIII-resistant DraI fragments of 3.7 and 1.8 kb, and the aberrant 5.4-kb DraI fragment is further rearranged.
Hygromycin-resistant NF clones retained at least one or more unaltered copies of the mutant hyg genes in 10/11 clones; 8/11 clones achieved hygromycin resistance via gene conversion of a single copy; 1/11 demonstrated an exchange event; 1/11 demonstrated an abnormal rearrangement and one exhibited hygromycin resistance without a clear etiology. By marked contrast, A-T clones retained unchanged sequences in 2/27 clones; 10/27 showed evidence of gene conversion; and 24/27 demonstrated complex rearrangements, in which hygromycin resistance was not a result of gene conversion or exchange in at least 17 of these 24 clones. The clone, C3, developed faint HindIII-resistant DraI bands, seen only on a later film, which suggested heterogeneity developing in the clone during the short period of amplification and presumably reflects the enhanced genetic instability of A-T cells. The clone W8 had no apparent hygromycin containing bands, despite keeping the cells in hygromycin until extracting DNA. Thawing the frozen cells from this clone revealed the cells to have re-developed hygromycin sensitivity. The suggestion is that intrinsic instability allowed the rapid development of the loss of hygromycin resistance genes. The intrinsic recombinational processes (and presumably repair) appear to be sequence altering in A-T and sequence conserving in normal cells. Rearrangements that fail to reconstitute a functional gene cannot be measured by these assays.
A-T cells show hypersensitivity to ionizing radiation which has been assumed to be due to a deficiency in DNA repair. In this paper, we have found A-T cells to have an abnormally high rate of recombination and to have abnormal, error-prone recombination in assays of chromosomal and extra-chromosomal recombination. The role of recombination in double-strand break repair and radiation sensitivity has been demonstrated recently in xrs cells (23) and scid cells(24, 25) . Both cell types have reduced or absent V(D)J recombination and a failure to close DNA double-strand breaks. By contrast, A-T cells have normal double-strand break closure but also demonstrate frequent chromosome aberrations(5, 26) . It is suggested that error-prone strand break processing may underlie the sensitivity to ionizing radiation in A-T.
The site of cleavage in relation to the sequences required to
recombine appears to be important. If the DNA termini are within or
close to the recombination substrates, then the high frequency of
recombination in A-T is offset by mis-repair of the recombining
sequences. Previous studies have found up to 5-fold differences in
recombination of extra-chromosomal plasmid DNA, which has been recorded
as not
significant(16, 27, 28, 29, 30) .
The results seen with co-transfection of KpnI-cleaved plasmid
could be due to extra-chromosomal rejoining between the 3` end of
p2 and the 5` end of p
3 rather than recombination. However,
the same results are obtained when only one of the two plasmids is
cleaved, or when p
2 is cleaved with EcoRV and p
3 is
cleaved with KpnI, which precludes a simple re-ligation
mechanism (data not shown). It is also concluded that A-T cells show
recombination mediated mis-repair around the site of strand breakage.
Recombination of vectors such
as p2/p
3 is generally thought to be extra-chromosomal, but
also requires an integration step. Recombination may occur by events
occurring extra-chromosomally, associated with integration, or
intra-chromosomally. Our data with p
2/p
3 and the previously
reported data cannot separate solely extra-chromosomal events from
events associated with integration, even though these assays have been
widely reported to reflect extra-chromosomal recombination. The assay
of V(D)J recombination evaluates entirely extra-chromosomal events, and
A-T cells have been shown to make functional coding and signal
joints(31) . However, this assay does not measure frequency or
fidelity of recombination separately, and may miss the error-prone
tendency of A-T cells because the assay detects only functional
rearrangements.