ABSTRACT
A novel DNA rearrangement has been characteriszed that is both a direct and inverted repeat. This rearrangement involves the 2-fold duplication of a plasmid sequence adjacent to the site of insertion of a long palindrome. The sequence of this rearrangement suggests that it has arisen by strand slippage from the leading to the lagging strand of the replication fork as a consequence of the presence of the long palindrome.
Genomes are not static, they have both the necessary levels of fidelity for
stable inheritance and the required flexibility for genetic change. A primary
mechanism of genetic change is recombination. Genome rearrangements in both
prokaryotes and eukaryotes may result from `homologous recombination', between
long homologous sequences (
1
-
3
), as well as from `illegitimate recombination', between sequences of little or
no homology (
2
,
4
).
Illegitimate recombination mechanisms are important in evolution (
5
,
6
), biotechnology (
7
,
8
) and have been suggested to play a role in repeated sequence instability
associated with certain human genetic disorders (
9
-
11
) and colorectal carcinomas (
12
,
13
). Illegitimate recombination may be divided into two broad classes: (i) end
joining, which is mediated by enzymes which cut and join DNA, such as
topoisomerases, site-specific DNases and proteins which initiate rolling circle replication;
(ii) strand slippage, where, after pausing at the replication fork, the nascent
strand can dissociate from one template and pair with another. Strand slippage
can occur in association with short homologous sequences, but, unlike
homologous recombination, this process is RecA independent both in plasmids (
14
-
16
) and the chromosome of
Escherichia coli
(
15
).
Sequences which have the potential to form unusual secondary structures are
known to promote pausing of DNA replication and can stimulate replication
slippage (
17
). Such sequences include: (i) oligopurine-oligopyrimidine sequences, able to adopt a triple helical structure in
supercoiled DNA, both
in vivo
(
18
) and
in vitro
(
19
); (ii) tri- and tetranucleotide repeats, thought to form pseudo-hairpins when single-stranded; (iii) alternating GC or GT dinucleotide repeats,
capable of forming Z-DNA; (iv) DNA palindromes (inverted repeats), able to form hairpins, when
single-stranded, or cruciforms, when double-stranded.
Long DNA palindromes are unstable in both eukaryotes and prokaryotes (
20
,
21
) and have been shown to halt the progress of the replication fork
in vitro
(
22
,
23
). DNA palindromes longer than 150-200 bp cannot be cloned in wild-type
E.coli.
Either the replicon containing the palindrome is so poorly replicated that it is
inviable (
24
,
25
) or the palindrome is so unstable that it suffers partial or complete deletion
(
26
,
27
). Many studies of deletion stimulated by palindromic sequences have shown that
their endpoints tend to occur in short direct repeats (
21
,
32
-
35
).
Escherichia coli
sbcCD
mutants are known to increase the viability of replicons containing long
palindromes (
28
-
31
) and this results in the stabilization of these sequences within a population.
In this work we have isolated a novel mutation composed of both direct and
inverted repeats that we call DIR (direct and inverted repeat). This
rearrangement is associated with loss of a 571 bp palindrome inserted at the
Eco
RI site in the multicloning region of plasmid pUC18. Its structure and origin
suggest that it may have arrisen by strand slippage across the replication
fork.
AXI medium is LB agar supplemented with 100 [mu]g/ml ampicillin, 64 [mu]g/ml X-gal and 30 [mu]g/ml IPTG. Plasmids were maintained in
E.coli
strains DL733 (JM83 [Delta]
sbcCD
::Km
R
) or JM83 (
36
) for sequencing of the DIR mutation. The 571 bp palindrome used in this work is
a perfect inverted repeat of 228 bp separated by 15 bp (
37
). It is composed of non-essential bacteriophage [lambda] DNA and was obtained from DRL 116 ([lambda] [Delta]
B pal spi
6
cI
857).
The plasmid vector used was pUC18 (
36
). The construction of pAC2, pMS7 and pDIR1 is described in Results. Plasmid DNA
was isolated using the Qiagen Plasmid Midi Kit (Qiagen Inc.) except for the
preparation of multimers, when DNA was isolated using the alkaline SDS method
of Birnboim and Doly (
38
).
Aliquots of 4 [mu]g plasmid DNA were digested to completion with 20 U
Eco
RI (Boehringer Mannheim) and radiolabelled using 10 [mu]Ci [[alpha]-
35
S]dATP (~600 Ci/mmol) and 1 U Klenow enzyme (Boehringer Mannheim). The DNA was
purified using the QIAquicktm Nucleotide Removal Kit (Qiagen Inc.). Samples of 2.5 [mu]g purified DNA were digested with 10 U
Bam
HI (Boehringer Mannheim) and purified once more using a QIAquicktm Nucleotide Removal Kit.
32
P-End-labelled marker V (Boehringer Mannheim) and marker 8-32 (Pharmacia) were used as size markers.
Radiolabelled samples were denatured by boiling and electrophoresed on a 10%
Longrangertm polyacrylamide gel (AT Biochem) containing 7 M urea. The gel was run at
55oC to prevent hairpin formation. After drying, the gel was autoradiographed.
Sequencing was carried out using the Sanger technique (
39
). Two primers were used: primer 5'-GACTGGAAAGCGGGCA-3' (596L) was manufactured by Perkin Elmer Econopuretm and primer 5'-GTTTTCCCAGTCACGAC-3' (-40) was
supplied with the Sequenase® v2.0 Sequencing Kit (US Biochemical). DNA was sequenced using the
Sequenase® v2.0 Sequencing Kit. DMSO sequencing was carried out with the Sequenase® v2.0 Sequencing Kit with the addition of 10% DMSO (Sigma Chemical
Co.) to the reaction mix and to the GATC termination mixes. Samples were
denatured by boiling and electrophoresed on a 6% Longrangertm polyacrylamide gel (AT Biochem) containing 7 M urea. The gel was run at
50oC and autoradiographed after drying.
A sample of 0.5-1 [mu]g plasmid DNA was digested to completion with 10 U
Hin
dIII or
Pvu
II (Boehringer Mannheim). Electrophoresis of undigested plasmid DNA was carried
out on 1% agarose gels (Flowgen) at 2 V/cm overnight.
Hin
dIII digests were carried out on 1% agarose gels (Flowgen) at 5 V/cm.
Electrophoresis of
Pvu
II digests was performed on 2% NuSieve
®
3:1 (Flowgen) agarose at 4 V/cm. Gels were stained with ethidium bromide (0.5 [mu]g/ml) and photographed using GRAB-ITtm (UVP Inc.) or Polaroid 667 film. A
Hin
dIII/
Eco
RI digest of [lambda] DNA or Marker VI (Boehringer Mannheim) was used as a size standard. In
peparation for sequencing, the
Pvu
II fragment containing the DIR insert was purified by electrophoresis, excised
from the gel and purified using a QIAquicktm Gel Extraction Kit (Qiagen Inc.).
Plasmid pAC2 was constructed by ligating a 571 bp DNA palindrome (
37
) into the
Eco
RI site of pUC18. When the
E.coli
strain
DL733 ([Delta]
sbcCD
::Km
R
) was transformed with pAC2 DNA the colonies obtained were observed to have an
unusual phenotype on AXI medium (white with blue sectors). This phenotype
suggested some genetic instability of pAC2, blue sectors being composed of
cells able to hydrolyse X-gal and white sectors being composed of cells unable to do so. Plasmid
pMS7 was isolated by plating DL733 containing pAC2 on AXI medium and looking
for rare white clones.
Plasmid DNA of pAC2 and pMS7 were compared by agarose gel electrophoresis. The
analysis of uncut pAC2 DNA revealed a population of different multimeric forms,
including a species whose migration suggested the existence of a heterodimer in
which only one copy of the palindrome was present (Fig.
1
a). Uncut pMS7 DNA was primarily a dimer of the palindrome-containing DNA (Fig.
1
a). This interpretation was supported by the DNA restriction pattern, after
digestion with
Hin
dIII (Fig.
1
b).
pAC2 and pMS7 were recircularized by digesting the plasmids with
Bsr
FI, religating the ends and transforming DL733 ([Delta]
sbcCD
). The results are shown in Table
1
. These results indicate a significant increase in the yield of highly sectoring
colonies derived from pMS7. Plasmid DNA was isolated from a number of these
sectoring clones (derived from both pAC2 and pMS7) and restriction analysis
revealed that although they had the sectoring phenotype of pAC2, they did not
contain the 571 bp DNA palindrome. One such plasmid, denoted pDIR1 (derived
from the monomerization of pAC2), was chosen for further detailed analysis.
Agarose gel electrophoresis of uncut pDIR1 DNA demonstrated that the simplest
form co-migrated with dimers of pUC18 (data not shown). However, restriction
analysis with
Pvu
II suggested that the plasmid was in fact predominantly a heterodimer of pUC18
and pUC18 with an insert of ~40 nt. Furthermore, this analysis suggested that the heterodimer present in
pAC2 DNA contained the 571 bp palindrome on one side and this same ~40 nt insert on the other (see Fig.
2
). This also confirmed that pMS7 was primarily present as homomultimers of
palindrome-containing DNA.
pAC2, pDIR1, pMS7 and pUC18 were digested with
Eco
RI and radiolabelled using [[alpha]-
35
S]dATP and Klenow enzyme. A sample of the purified DNA was digested with
Bam
HI, run on a 10% Longrangertm polyacrylamide gel and the bands visualized by autoradiography after the
gel had dried (Fig.
3
). The restriction pattern obtained confirmed the presence of a second
Eco
RI site in the multicloning region, giving a 46 bp fragment (42 bp plus 4 bp
derived from filling in the
Eco
RI site overhang with Klenow enzyme) in both pAC2 and pDIR1. pMS7 and pUC18 do
not have this second
Eco
RI site. In contrast, all four plasmids have the correct 25 bp (21 plus 4 bp)
Eco
RI-
Bam
HI fragment.
DNA sequencing of the polylinker region of pDIR1, pUC18 and pAC2 was carried out
on both strands using two primers: 596L and -40 (see Materials and Methods). Since pDIR1 and pAC2 were found to
contain heteromultimers, sequencing of the 42 bp insert required its separation
from the other component of the multimer. This was done in two ways. Firstly,
the
Pvu
II fragment containing the insert was purified from the other parts of the
multimer by agarose gel electrophoresis and sequencing was performed on the DNA
fragment isolated from the gel. Secondly, pDIR1 DNA was used to transform JM83
and a homomultimer containing the 42 bp insert was isolated that could be
sequenced directly. Normal `Sanger' DNA sequencing revealed a region in the
multicloning site where the DNA polymerase seemed to stall. This suggested the
presence of secondary structure. Sequencing in the presence of 10% DMSO
improved the ability of the polymerase to sequence through this region and
revealed the presence of a novel type of mutation (see Fig.
4
). The sequence shows the presence of a duplicated
Eco
RI-
Bam
HI multicloning site fragment with the same region inverted in between the
duplication. We call this a `DIR' structure, since it is composed of both
direct and inverted repeats.
A 571 bp imperfect palindrome of bacteriophage [lambda] DNA was introduced into the cloning vector pUC18 to form plasmid pAC2
and an unstable phenotype was observed. It was initially thought that the
unstable phenotype of pAC2 might be due to deletion of the 571 bp palindrome.
Deletion of the palindrome would allow production of a functional [alpha] subunit of [beta]-galactosidase.
Escherichia coli
cells harbouring the plasmid would have a dark blue colour (the result of
hydrolysis of X-gal, a chromogenic substrate) and this would generate a sectoring clone.
However, this proved not to be the case. A derivative of pAC2 was isolated that showed almost no sectoring and this plasmid (pMS7) still contained the palindrome. Furthermore, when pAC2 and pMS7 were monomerized sectoring clones were discovered that did not contain the 571 bp DNA palindrome. One of these clones was analysed in detail and was found to contain an inverted and directly duplicated region of the plasmid polylinker adjacent to the site of insertion of the palindrome, but the palindrome itself was no longer present. We have called this structure a DIR for `direct and inverted repeat'. The same DIR structure was also found to be present in pAC2, which is a population of DNAs including a heteromultimer composed of one monomeric subunit containing the palindrome and the other containing the DIR structure. The stabilized plasmid pMS7 was found to be primarily a homomultimer of the palindrome-containing subunit. The unstable (sectoring) derivatives of pAC2 and pMS7 that had lost the 571 bp palindrome were all found to be heteromultimers with one subunit containing the DIR structure and the other containing wild-type pUC18 sequence. Small amounts of a DNA fragment of the predicted size of pUC18 were also seen in the DNA of pAC2. Since all these plasmids were maintained in Rec+ cells, interconversion between various forms was occurring to generate subpopulations of plasmids. The results shown in Table 1 are consistent with heterogeneous populations of plasmids in the DNA preparations of pAC2 and pMS7 and the isolation of subpopulations by forcing monomerization. Alternatively, monomerization may stimulate rearrangement and future work will be needed to distinguish between these two possibilities. The plasmids investigated here are shown diagrammatically in Figure 5 .
We thank Alison Chalker for construction of plasmid pAC2, Morgan Shaw for
isolation of pMS7 and Thorsten Allers for photography. Funding for this work
has come from the BBSRC, the MRC and NBL Gene Sciences Ltd.
Plasmid
%Total
Blue colonies
Sectoring colonies `pAC2 like'
White colonies
pAC2 cut with
BsrFI+ligase
5.5
86
8.5p
MS7 cut with BsrFI+ligase
0
8.5
91.5
pAC2 cut with BsrFI-ligase
9
75
15
pMS7 cut with BsrFI-ligase
0
0
0
pAC2 uncut
10.5
80
9.5
pMS7 uncut
0
1
99
REFERENCES
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