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INTRODUCTION |
The genetic instability of three triplet repeat sequences
(TRS),1 CTG·CAG, CGG·CCG,
and TTC·GAA, has been shown to result in approximately 12 hereditary
neurological diseases (1-3) including myotonic dystrophy (4),
Kennedy's disease (5), fragile X disease (6), and Freidreich's ataxia
(7). These diseases are inherited in a non-Mendelian fashion by a
phenomenon called "anticipation," which is characterized by an
increase in severity and a decrease in age of onset from one generation
to the next. The anomalous expansion of TRS has been identified as the
molecular basis for anticipation (1). Triplet repeat tracts are highly
polymorphic and have been shown to range from five to approximately 40 repeats in normal human chromosomes. Expansion of these tracts can
result in type I and type II diseases as classified by Paulson and
Fischbeck (3). Whereas type I diseases are characterized by modestly expanded TRS (approximately 30-80 repeats) in the coding region of a
gene, type II diseases contain massive expansions (>1000 repeats) of
the triplet repeat tract in the 5'-UTR or 3'-UTR or in an intron of a
gene (1, 3).
Escherichia coli has been used as a genetically tractable
system for the study of TRS in vivo (8). The genetic
instability of TRS in E. coli is dependent on its
orientation relative to the unidirectional ColE1 origin of replication
as well as host cell growth phase and transcription (9-13). Other
factors including genetic background (12), methyl-directed mismatch
repair (14), and expression of single-stranded DNA binding protein
(SSB) (15) are also important. The effect of the orientation of the TRS
with respect to the direction of replication on its instability was also demonstrated in Saccharomyces cerevisiae (16-18).
Single-stranded phage replication from the filamentous f1 and M13 viral
origins was characterized both in vitro and in
vivo (19-21). In these phages, the unidirectional synthesis of
the Watson and the Crick strands of the DNA takes place by different
mechanisms. First, the single-stranded (+)-strands are converted to the
double-stranded replicative form (RF) by complementary strand
synthesis. Second, the RF is replicated by a rolling circle mechanism
to yield (+)-strands. Remarkably, both steps occur by continuous strand
synthesis and are marked by the absence of discontinuous Okazaki
fragments. Therefore, due to the absence of any step analogous to
lagging strand synthesis, rolling circle replication has been exploited in in vitro replication studies as a model to simulate the
synthesis of the leading strand (22, 23).
We proposed (9) that the in vivo expansion and deletion of
the CTG·CAG repeats is mediated by hairpin formation by the CTG
repeats on the lagging strand during DNA replication. This model was
based on two findings. First, single-stranded TRS have been shown
(24-28) to adopt compact secondary structures in vitro (see
below). Second, replication-dependent deletions between
direct repeats occur due to secondary structure formation
preferentially on the discontinuous lagging strand (29), because the
lagging strand template is more likely to transiently be in a
single-stranded state compared with the leading strand. However, the
role of the leading strand in deletion formation has not been well
studied. Kang et al. (9) could not distinguish between TRS
instabilities on the leading strand from those on the lagging strand,
because the two strands are synthesized concurrently in the ColE1
replication system. Therefore, we utilized the filamentous phagemid
system (30) to dissect the replication fork and focus on the continuous leading strand synthesis of TRS in vivo.
A substantial body of evidence (25, 31, 32) indicates that the genetic
instability of TRS is derived from their intrinsic biophysical
properties. In vitro studies on short single-stranded oligonucleotides containing TRS showed their capacity to adopt compact
secondary structures (25, 27, 28) including hairpin loop conformations
(33-37), tetraplexes (38, 39), and slipped structures (40, 41). Short
single-stranded tracts containing CTG repeats have a higher propensity
to form hairpin structures than similar tracts containing the
complementary CAG repeats (33, 34, 37), possibly accounting for the
orientation-dependent behavior of these repeats in
replication (9). The ability of the CGG repeats to form secondary
structures in vitro also differs significantly from the
complementary CCG repeats (25, 27). Whereas short CGG repeats form
hairpins (42, 43) or tetraplexes (38, 39), the CCG repeats exclusively
form hairpins (36, 44, 45). However, there is not a consensus in the
literature regarding the relative stabilities of the structures formed
by the CCG and the CGG repeats. The secondary structures formed by the
TRS tracts have been shown to impede the progression of DNA polymerases
in vitro (39, 46, 47) and in vivo (48).
Herein, we show that instability of the CTG·CAG repeats in an
in vivo single-stranded phage replication system depends on the orientation of the repeats with respect to the f1 replication origin. Substantial deletions were observed when the CTG repeats that
are prone to form hairpins are present in the template for rolling
circle replication. Because f1 replication is characterized by the
absence of a discontinuous lagging strand, our data show that deletion
of TRS tracts can occur on the leading strand of DNA replication in
E. coli. We also observe expansion of CGG·CCG and
CTG·CAG repeats during complementary strand synthesis in
vivo.
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EXPERIMENTAL PROCEDURES |
Strains and Helper Phage--
E. coli host strain
NM522 (F' lac Iq
(lacZ) M15
proA+B+/supE thi
(lac-proAB)
(hsdMS-mcrB)5
(rk
mk
McrBC
))
and helper phage M13K07 (KanR) were purchased from Promega
Corp. E. coli NM522 is proficient for homologous recombination.
Cloning Vectors, Oligonucleotides, and Probes--
The phagemid
cloning vectors pGEM3Zf+ and pGEM3Zf
(3199 bp), which carry the f1
origin of replication in addition to the ColE1 origin, were purchased
from Promega Corp. The f1 origin is in opposite orientations in
pGEM3Zf+ and pGEM3Zf
. Sequencing of single and double-stranded DNA
was done using either the T7 promoter primer
(5'-TAATACGACTCACTATAGGG-3') or the SP6 promoter primer
(5'-TATTTAGGTGACACTATAG-3') purchased from Promega Corp. Southern
hybridization was done using a CTG repeat containing probe
5'-GATA(CTG)15-3' (Gene Technologies Laboratory, Texas A & M University).
Cloning of CTG·CAG, CGG·CCG, and TTC·GAA Inserts into
pGEM3Zf+ and pGEM3Zf
Vectors--
Fragments containing CGG·CCG and
CTG·CAG TRS were prepared from previously described plasmids (9, 12)
by digesting 10 µg of plasmid DNA with HindIII and
SacI (New England Biolabs Inc.). Fragments containing
TTC·GAA triplet repeats were generated by digesting pRW3804 with
EcoRI and PstI (New England Biolabs Inc.). The
digested DNA was electrophoresed on a 5% acrylamide gel, and the band
containing the triplet repeat fragment was excised. The DNA was eluted
from the excised band and purified by phenol extraction. The vector was
prepared by digesting pGEM3Zf+ and pGEM3Zf
with either
HindIII and SacI or EcoRI and
PstI. The linearized vector was electrophoresed on a 1%
agarose gel, and the DNA eluted from the excised band. The vector DNA
was dephosphorylated with calf intestinal alkaline phosphatase
(Boehringer Mannheim). The insert and vector were mixed and ligated for
16 h at 16 °C by the addition of 1 unit of T4 DNA ligase (U.S.
Biochemical Corp.). The ligation mixture was transformed into E. coli NM522 by electroporation. Plasmid DNA was isolated from
individual transformants by standard alkaline lysis procedures. The DNA
was characterized by restriction mapping and dideoxy chain termination
sequencing of the insert in both strands. The sequences cloned and
characterized are listed in Table I.
Cloning of (CAG·CTG)175 in Orientation II--
The
CAG·CTG inserts were prepared by digesting 10 µg of pRW3711 (Table
I) with EcoRI and BspMI. The DNA was then blunt ended by
filling in the cohesive ends with 10 units of the Klenow fragment of
E. coli DNA polymerase I (U.S. Biochemical Corp.) and dNTPs. The insert was then electrophoresed on a 5% acrylamide gel, excised, and eluted as described above. The vector was prepared by digesting pGEM3Zf+ and pGEM3Zf
with EcoRI and BspMI,
followed by filling in the cohesive ends as described above. The
linearized, blunt ended vector was purified by elution from a 1%
agarose gel. The vector and insert were mixed and ligated by the
addition of 10 units of T4 DNA ligase for 16 h at 16 °C. The
ligation mixture was transformed into E. coli NM522 by
electroporation. Plasmid DNA was isolated from individual transformants
and characterized by restriction mapping and sequencing of both insert strands.
Purification and Characterization of Single-stranded
DNA--
E. coli NM522 was transformed with the appropriate
plasmid by electroporation and plated on LB Agar plates (1%
bacto-tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing
100 µg/ml ampicillin. Single transformant colonies were inoculated
into 10 ml of LB (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl)
and grown at 37 °C with shaking at 250 rpm. When the cells had grown
to an absorbance (600 nm) of 0.2 units, the tubes were inoculated with helper phage M13K07 at a concentration of 1 × 108
plaque-forming units/ml. The cells were further grown at 37 °C with
shaking at 250 rpm for 2 h. The cultures were then inoculated into
flasks containing 1 liter of LB containing 100 µg/ml ampicillin and
100 µg/ml kanamycin (for maintenance of M13K07) and grown at 37 °C
with shaking at 250 rpm for 12 h. The cultures were centrifuged in
a Sorvall RC3Bplus centrifuge at 2500 × g for 20 min,
and the supernatant containing the packaged phagemids was collected.
The single-stranded phagemid DNA was isolated and purified from the packaged phagemids according to previously described procedures (49).
The single-stranded DNA was then characterized by standard dideoxy
chain termination sequencing (50). Control cultures of E. coli NM522 uninfected by M13K07 were unable to grow in the presence of 100 µg/ml kanamycin, thus validating the use of kanamycin selection for the continued presence of M13K07.
Propagation of Triplet Repeat-containing Phagemids in E. coli--
Phagemids containing undeleted TRS tracts were prepared as
described previously (10) and transformed into competent E. coli NM522 cells, which were preinfected with M13K07 helper phage
and maintained by kanamycin selection. The transformation mixture was
inoculated into 10-ml LB tubes (containing ampicillin and kanamycin,
both at 100 µg/ml) at a cell density of 103 cells/ml. The
cultures were grown at 37 °C with shaking at 250 rpm. When the
cultures reached an absorbance (600 nm) of 1.0 unit (20-24 h), an
aliquot was inoculated into a fresh tube of 10 ml LB (with kanamycin
and ampicillin as before) at a final dilution of 1 × 10
7. The original culture was harvested, and
double-stranded RF DNA was isolated by standard alkaline lysis
procedures (50). The cultures were thus maintained in log phase growth
by repeated recultivation (defined in the figure legends as number of
recultivations). The phagemid RF DNA from the harvested cells was
analyzed for the stability of its triplet repeat tract. Since the
phagemid life cycle involves the formation of the RF from the
(+)-strand, we assume that this analysis is an accurate measure of the
stability of the triplet repeat tract in the (+)-strand. However, we
cannot exclude the formal possibility that aberrant DNA molecules are produced that are not amplified by plasmid replication and hence are
not detected.
Southern Blot and Polyacrylamide Gel Analysis of Triplet Repeat
Instabilities--
The triplet repeat insert was excised from the
phagemids with EcoRI and HindIII and analyzed as
follows. In order to identify the deletion products, Southern
hybridization was performed on pRW3711 and pRW3712 digested with
EcoRI and HindIII and separated on 1.5% agarose
gels. The gels were blotted by standard procedures (50) and probed with
a CTG-containing oligonucleotide that was 5'-end-labeled with
[
-32P]dATP and T4 polynucleotide kinase under
conditions of medium stringency (50% formamide, 50 °C for 6 h). The blots were washed, dried, and exposed to x-ray film. The extent
of triplet repeat instability was determined by measuring the relative
amount of undeleted triplet repeat insert in the phagemids after
replication in the presence of M13K07. The
EcoRI-HindIII-digested DNA was labeled by end
filling with the Klenow fragment of DNA polymerase I and
[
-32P]dATP. The labeled DNA was separated on 5%
polyacrylamide gels, which were dried and exposed to a
phosphorescence-sensitive screen. The instability was quantitated by
scanning the exposed screen with a Molecular Dynamics PhosphorImager.
The amount of radioactivity (as estimated by the signal intensity) in
the band corresponding to the full-length TRS was measured as a
proportion of the total radioactivity in the lane below the band.
 |
RESULTS |
Instability of (CTG·CAG)175 Depends on the
Orientation of the Sequence with Respect to the f1 Origin of
Replication--
In vivo growth of plasmids containing
CTG·CAG triplet repeats in E. coli has revealed the
involvement of DNA replication in the instabilities associated with
these sequences (9). It was hypothesized that expansions and deletions
occur due to formation of hairpin structures by CTG repeats on the
lagging strand template (28). This was based on the earlier finding
that the discontinuous synthesis of Okazaki fragments on the lagging
strand increases its probability of existing in a single-stranded state
relative to the leading strand template, thereby resulting in
preferential mutagenesis of the lagging strand (29). The replication of
these sequences in a system where leading and lagging strands can be dissected would therefore be an attractive way to test this hypothesis. Mechanistically, the replication from the filamentous phage f1 origin
can be distinguished into two stages (19-21). First, the single-stranded (+)-strand template is converted to double-stranded RF
by a continuous complementary strand synthesis step. Second, the
double-stranded RF is replicated via a rolling circle replication step
to yield single-stranded (+)-strands. The rolling circle replication is
also continuous and is analogous to leading strand synthesis (22, 23).
The absence of a discontinuous strand synthesis step involving Okazaki
fragments is a characteristic feature of replication from the f1 origin
(51).
In order to delineate the instabilities that occur due to the formation
of hairpin structures during rolling circle replication (and therefore,
by extension, during leading strand synthesis) from those that occur
due to similar structures during complementary strand synthesis, we
established an in vivo phagemid replication system for TRS.
The phagemids pGEM3Zf+ and pGEM3Zf
were used, which carry the origin
of replication from the filamentous phage f1 oriented oppositely in the
two phagemids. This facilitates the replication of the top strand by
pGEM3Zf
and the bottom strand by pGEM3Zf+ when rolling circle
replication is induced. Replication from the f1 origin can be induced
by the use of a helper phage such as M13K07, which infects E. coli host strains that have conjugal F pili (52). The host strain
NM522 was chosen because it carries an F' factor and thus has the F
pilus (53). When grown in E. coli NM522 in the presence of
helper phage M13K07, the phagemids are replicated from the f1 origin by
rolling circle and complementary strand synthesis (54).
pRW3711 and pRW3712 (Fig. 1) contain
(GCT)27ACT(GCT)40ACT(GCT)106
(referred to as (CTG·CAG)175 for convenience) cloned into the polylinker of pGEM3Zf+ and pGEM3Zf
, respectively. We propagated phagemids pRW3711 and pRW3712 in log phase in E. coli NM522
in the presence of the helper phage M13K07 as described under
"Experimental Procedures." To confirm that the phagemids were being
replicated from the f1 origin, the single-stranded (+)-strands were
purified from the supernatant and characterized by dideoxy chain
termination sequencing (data not shown). The E. coli NM522
cultures carrying pRW3711 and pRW3712 were maintained in log phase
growth by repeated recultivation. After each recultivation, the
cultures were harvested, and the double-stranded phagemid DNA were
isolated. The DNAs were digested, end-labeled, and analyzed on 1%
agarose and 5% polyacrylamide gels. The agarose gels were blotted onto
nylon membranes and hybridized to a radiolabeled probe containing 15 repeats of CTG in order to verify the identities of the putative
deletion products. It was observed that the CTG-containing probe
hybridized to the band containing the full-length TRS as well as to the
bands of shorter lengths (data not shown). An analysis of the deletion
products was performed by electrophoresing the digested DNA on
polyacrylamide gels (Fig.
2A).

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Fig. 1.
Orientation of (CTG·CAG)175 in
recombinant phagemids. Phagemids pRW3711 and pRW3712 (R. P. Bowater and R. D. Wells, unpublished data) contain
(CTG·CAG)175 in the same orientation (orientation I) with
respect to the ColE1 origin and in opposite orientations with respect
to the f1 origin. When replication is initiated at the f1 origin,
pRW3711 yields a plus strand that contains CAG repeats, whereas pRW3712
yields a plus strand that contains CTG repeats.
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Fig. 2.
In vivo instability of
(CTG·CAG)175 and (CTG·CAG)130.
Phagemids containing (CTG·CAG)175 (A) or
(CTG·CAG)130 (B) were isolated from E. coli NM522 cultures after repeated recultivations of log phase
growth. The DNA was digested with EcoRI and
HindIII, labeled with [ -32P]dATP, separated
on 5% acrylamide gels, and exposed to x-ray film. The lanes
numbered 1-5 contain DNA isolated from cultures
harvested after 1-5 recultivations. The arrow indicates the
band containing the full-length triplet repeat. The deletion products
migrate in the region encompassed by the open
box. All (CTG·CAG)n-containing restriction
fragments also contain 116 bp of nonrepetitive flanking sequence.
A shows the analysis of the deletion products from the
growth of pRW3711 and pRW3712, which contain
(CTG·CAG)175. The difference in the electrophoretic
mobilities of the TRS-containing fragments is due to the difference in
size (see legend to Table I). B shows a similar analysis of
deletion products from the growth of pRW3111 and pRW3121, which carry
(CTG·CAG)130. C, the extents of the
instabilities of (CTG·CAG)130 and
(CTG·CAG)175 were measured by exposing the dried 5%
acrylamide gels from the recultivation experiments with pRW3111 ( )
(top curve), pRW3121 ( ) (next
to top curve), pRW3711 ( ), and
pRW3712 ( ) (shown in Fig. 2, A and B) to a
Molecular Dynamics PhosphorImager screen followed by scanning. The
amount of radioactivity (as estimated by the signal intensity) in the
band corresponding to the full-length TRS was measured as a proportion
of the total radioactivity in the lane below the
band. This was taken as the percentage of molecules in the
sample that contained an undeleted TRS tract. The percentage of
undeleted TRS was plotted on the y axis against the number
of recultivations. The data were computed as an average of three
separate experiments. The error bars indicate the
S.D. values. The curves were drawn by connecting the points manually on
the program Canvas 5.0 (Deneba Software Inc.) using the Beziér
curve tool.
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Upon quantitation of the deletion products, a substantial difference
was observed in the stability of the TRS between pRW3711 and pRW3712 in
the presence of helper phage M13K07 (Fig. 2C). In the case
of pRW3711, the full-length (175 repeats) TRS had been completely
deleted to a TRS tract 20-30 repeats in length by the third
recultivation. On the other hand, the TRS tract in pRW3712 had up to
55% undeleted full-length TRS remaining even after five recultivations
in log phase. When pRW3711 and pRW3712 were propagated in E. coli NM522 in the absence of M13K07 helper phage, there was no
significant deletion of their TRS tracts in both cases even after five
recultivations (data not shown).
These experiments show that the difference in stability between pRW3711
and pRW3712 are dependent on the f1 origin, because the two plasmids
are identical in all respects except for the orientation of the f1
origin. Furthermore, since the instabilities are observed only in the
presence of helper phage, replication from the f1 origin is required
for the stability differences in the TRS tracts between these two plasmids.
Instability of (CTG·CAG)n Depends on the Length of
the TRS Tract--
The severity and the lower age of onset of triplet
repeat diseases have been correlated with an increase in the length of the TRS tract in certain genes in patients (1-3, 28, 55). The
biochemical properties of CTG·CAG such as their ability to bind
nucleosomes (56, 57), to block the progression of DNA polymerases (46,
47), and to circularize due to their inherent flexibility (58, 59) have
been shown to be dependent upon their length. Furthermore, the
stability of CTG·CAG in plasmids in E. coli is also
dependent on the length of the repeat tract (9-11, 60). In order to
determine the effect of CTG·CAG length on their genetic instability
in our phagemid replication system, plasmids pRW3111 and pRW3121 (46)
were used, which contain (GCT)27ACT(GCT)102 (referred to as (CTG·CAG)130 for convenience) cloned into
pGEM3Zf+ and pGEM3Zf
, respectively (Table
I). These plasmids were propagated in
E. coli NM522 in log phase, and the RF DNA was isolated
after each recultivation. The CTG·CAG tract was excised from the RF DNA with EcoRI and HindIII, end-labeled with
[
-32P]dATP, and electrophoresed on 5% polyacrylamide
gels (Fig. 2B). Quantitation of these gels (Fig.
2C) showed that over the period of five recultivations,
there was no significant difference in the stabilities of the repeat
tracts of pRW3111 and pRW3121. In contrast to the
(CTG·CAG)175 tract in pRW3711 and pRW3712, the (CTG·CAG)130 tract in pRW3111 and pRW3121 showed the
following properties. First, the (CTG·CAG)130 tract was
almost completely stable (<10% formation of deletions) even after
five recultivations when replicated from the f1 origin. This is in
sharp contrast to the instability observed in the case of pRW3711 and
pRW3712. Second, there was no difference between the stabilities
observed for pRW3111 and pRW3121, as observed for pRW3711 and pRW3712. This clearly shows that the in vivo processes responsible
for the deletion of the (CTG·CAG)175 tract do not
destabilize the shorter (CTG·CAG)130 tract. Thus, the
length of the CTG·CAG tract determines its instability in this
filamentous phage replication system.
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Table I
Phagemids containing triplet repeats
Inserts containing triplet repeat sequences of different lengths were
cloned into the phagemid vectors pGEM3Zf+ and pGEM3Zf . Orientation is
defined with respect to the double-stranded unidirectional ColE1 origin
of replication. Hence, sequences in orientation I have the CAG, CCG, or
TTC tracts comprising the lagging strand template. Sequences in
orientation II have CTG, CGG, or GAA tracts comprising the lagging
strand template. The number of repeats in the (GAA·TTC)150
and (CTG·CAG)175 tracts has an error of ±5 repeats. In all
other cases, the exact repeat number has been determined by DNA
sequencing. Replication of the phagemids from the f1 origin was
confirmed by the purification of single-stranded DNA from the phagemid
particles in the culture supernatant. The single-stranded DNA was
characterized by sequencing.
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Reversing the Orientation of (CTG·CAG)n in the
pGEM3Zf+ and pGEM3Zf
System Results in the Reversal of the
Instabilities--
The differences observed between the stabilities of
pRW3711 and pRW3712 could be attributed to the fact that the rolling
circle template was composed of CTG repeats in pRW3711 and CAG repeats in pRW3712. Therefore, hairpins formed by CTG in the rolling circle template could be bypassed during DNA replication, thus resulting in
substantial deletion formation. However, there was also the possibility
that derivatives of pGEM3Zf+ (pRW3711) were more unstable than
derivatives of pGEM3Zf
(pRW3712) due to an inherently greater instability of sequences cloned in pGEM3Zf+ than those cloned in
pGEM3Zf
. In order to conclusively show that the deletions are
directed not by the vector sequence but by the sequence of the triplet
repeat that forms the template strand (CTG or CAG), we inverted the
orientation of the triplet repeat tract in pGEM3Zf+ and pGEM3Zf
. Upon
the inversion of the triplet repeat orientation, the rolling circle
template of the pGEM3Zf+ derivative would contain the CAG repeats,
while that of the pGEM3Zf
derivative would contain the CTG repeats.
We hypothesized, therefore, that the pGEM3Zf+ derivative would be more
stable in the in vivo filamentous phage replication system
than the pGEM3Zf
derivative.
Hence, we constructed phagemids pRW3539 and pRW3540 (Fig.
3), where the (CAG·CTG)175
tract was cloned into pGEM3Zf+ and pGEM3Zf
, respectively, in
orientation II (see "Experimental Procedures"). The phagemids were
then propagated in E. coli NM522 in log phase for five
successive recultivations, and the RF DNA was analyzed by restriction
digestion, end labeling, and electrophoresis through 5% polyacrylamide
gels (Fig. 4A). Upon
quantitation of these gels, it became evident that pRW3540 is more
unstable than pRW3539 (Fig. 4B) when replicated from the f1
origin. Thus, while the (CTG·CAG)175 tract in
orientation I is less stable in pRW3711 than in pRW3712 (Fig. 2), the
(CAG·CTG)175 tract in orientation II is more stable in
pRW3539 than in pRW3540 (Fig. 4). Thus, a reversal of the orientation of the CTG·CAG tract relative to the f1 origin results in the reversal of the instabilities.

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Fig. 3.
Orientation of (CAG·CTG)175 in
recombinant phagemids. Phagemids pRW3539 and pRW3540 contain
(CAG·CTG)175 cloned in the opposite orientation
(orientation II) compared with pRW3711 and pRW3712 (Fig. 1). Both
pRW3539 and pRW3540 contain the TRS in the same orientation (II) with
respect to the ColE1 origin but in opposite orientations with respect
to the f1 origin. When replication is initiated at the f1 origin,
pRW3539 yields a plus strand that contains CTG repeats, whereas pRW3540
yields a plus strand that contains CAG repeats.
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Fig. 4.
In vivo instability of
(CAG·CTG)175. A, pRW3539 and pRW3540 were
analyzed as in the legend to Fig. 2A. B, the
instabilities of pRW3539 ( ) and pRW3540 ( ) were quantitated as
described in the legend to Fig. 2C from three separate
recultivation experiments. The data treatment is also as described in
the legend to Fig. 2C.
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This experiment clearly shows that the instability of the repeat tract
in this filamentous phage replication system is determined by the
triplet repeat composition of the template strand during rolling circle
and complementary strand synthesis stages. The CTG repeat has a higher
propensity to adopt fold-back structures than the CAG repeat (33, 34).
However, methodology does not exist to enable the analysis of hairpin
loops in vivo (25). In this replication system, instability
of the CTG·CAG tract is high when the CTG tract is the template
during rolling circle replication. Therefore, our data implicate the
rolling circle step of the phagemid replication cycle to be where
fold-back structures are formed by the CTG repeat. This results in the
deletion of the CTG·CAG repeat tract observed in pRW3711 and pRW3540.
Instabilities of (CGG·CCG)81 Depend on the
Orientation of the f1 Origin and on the Length of the Repeat
Tract--
In addition to the CTG·CAG repeats, the instabilities of
the CGG·CCG repeats (12) and the TTC·GAA repeats (13, 61) have been
shown to be dependent on their orientations relative to the ColE1
unidirectional origin of duplex plasmid replication. Shimizu et
al. (12) propagated CGG·CCG repeat tracts cloned in pUC19 in a
variety of E. coli host strains and observed a clear effect of the orientation of the repeats relative to the origin of replication on their instability. They showed that deletions predominate when the
CGG repeat occurs on the template of the lagging strand. This is
consistent with the in vitro evidence that CGG repeats can adopt fold-back structures (43).
In order to determine if the f1 orientation affects the stabilities of
these sequences, we investigated the properties of pRW3517 and pRW3518
(Table I) in the filamentous phage replication system. These phagemids
were constructed by cloning a tract of (CGG·CCG)81
originally from the human FRAX A gene from patients with the
fragile X disease (12) into pGEM3Zf+ and pGEM3Zf
. The two phagemids
were propagated in E. coli NM522 in log phase for five
recultivations in the presence of helper phage M13K07. The RF DNA was
isolated and analyzed as before, and the analysis is shown in Fig.
5A. The quantitation of the
instabilities revealed that the (CGG·CCG)81 tract was
substantially more unstable in pRW3517 (Fig. 5B) than in
pRW3518 (Fig. 5C). Whereas by the fifth recultivation, less
than 5% of the (CGG·CCG)81 tract remained undeleted in
pRW3517, as much as 42% of the TRS in pRW3518 was undeleted at the
same stage. After six recultivations, there was no detectable undeleted
TRS in pRW3517, but 30% of the TRS was undeleted in pRW3518. Thus, the
orientation of the f1 replication origin has a substantial influence on
the stability of the (CGG·CCG)81 tract. Furthermore, the
phagemid pRW3517 contains the CGG repeat in the minus strand, which
forms the template for rolling circle replication. It is therefore
likely that the CGG repeats form fold-back structures on the minus
strand during rolling circle replication, resulting in massive deletion
of the TRS tract.

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Fig. 5.
In vivo instability of
(CGG·CCG)81. A, pRW3517 and pRW3518
(Table I) were analyzed as described in the legend to Fig.
2A. All (CGG·CCG)81-containing restriction
fragments also contain 76 bp of nonrepetitive flanking sequence.
B, the instabilities of pRW3517 were quantitated as follows.
The extents of the instabilities of (CGG·CCG)81 were
measured by exposing the dried 5% acrylamide gels from the
recultivation experiments with pRW3517 (shown in Fig. 5A) to
a Molecular Dynamics PhosphorImager screen followed by scanning. The
amount of radioactivity (as estimated by the signal intensity) in the
band corresponding to the (CGG·CCG)81 tract ( ) was
measured as a proportion of the total radioactivity in the lane below
the band. This was taken as the percentage of molecules in the sample
that contained an undeleted TRS tract. The relative amounts of each of
the deletion products of (CGG·CCG)81 were also determined
by measuring the signal intensity for a band as a proportion of the
cumulative signal intensity for all the deletion products in the lane.
The deletion products that constituted >4% of total TRS at any stage
during the growth were identified and plotted. The percentage of total
TRS constituted by each deletion product was plotted on the
y axis against the number of recultivations. The
curves were drawn as in Fig. 2C. The deletion
products identified from pRW3517 were (CGG·CCG)20 ( )
and (CGG·CCG)6 ( ). C, the instabilities of
pRW3518 were quantitated as described above. The data treatment is also
as described above. The full-length TRS tract containing
(CGG·CCG)81 is represented by . The deletion products
identified from pRW3518 were (CGG·CCG)52 ( ),
(CGG·CCG)45 ( ), (CGG·CCG)39 ( ),
(CGG·CCG)34 ( ), and (CGG·CCG)27 ( ).
We estimate that the product sizes are ±1 repeat unit.
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The instability of the CGG·CCG repeats is dependent on the length of
the tract in fragile X patients (6). We showed previously (12) that
CGG·CCG repeat tracts are unstable when cloned in plasmids in
E. coli in a length-dependent manner. Short
tracts containing up to 10 repeats were completely stable, whereas
tracts containing more than 24 repeats were increasingly unstable when cloned in pUC19 and propagated in E. coli DH5
. Therefore,
we studied the effect of the length of the CGG·CCG repeat on its instability in the filamentous phagemid replication system in vivo. pRW3511 and pRW3512 (Table I) were constructed by cloning (CCG·CGG)6 into pGEM3Zf+ and pGEM3Zf
, respectively.
Characterization of the double-stranded RF after propagation in
E. coli NM522 in the presence of M13K07 over five
recultivations revealed that the TRS in both pRW3511 and pRW3512 was
highly stable (>95% undeleted) even after five recultivations (data
not shown). Also, there was no f1 orientation-dependent
difference in the stabilities of pRW3511 and pRW3512. This behavior of
the short CGG·CCG tracts is in agreement with the observations of
Shimizu et al. (12).
TRS Deletion Occurs Predominantly during Rolling Circle
Replication--
We have observed a substantial influence of
replication from the f1 origin on the genetic instability of CTG·CAG
and CGG·CCG repeat sequences. For the CTG·CAG repeats (Fig. 1),
pRW3711 contains CTG in the (
)-strand and is genetically less stable
than pRW3712, wherein CAG is in the (
)-strand (Fig. 2C).
For the CGG·CCG repeats (Table I), pRW3517, in which CGG is in the
(
)-strand, is more unstable (Fig. 5B) than pRW3518, which
carries CCG in the (
)-strand (Fig. 5C). The greater
instability of the TRS tracts in the phagemids that contain CTG and CGG
repeats in the (
)-strand rolling circle template suggests the
following. First, in vitro experiments have shown (33, 34,
37) that CTG repeats have a higher propensity to form hairpins than CAG
repeats. Therefore, we propose that the rolling circle template is the
location for the formation of hairpins by the CTG repeats, resulting in
substantial deletions (Fig. 6,
left). Also, based on in vivo observations,
several workers have suggested (12, 48, 62) that CGG repeats of
physiologically relevant lengths are more likely to form fold-back
structures than CCG repeats. Hence, it can be concluded that the
deletions of the CGG·CCG repeats also take place on the rolling
circle template due to CGG-containing hairpins. Second, the full-length
(CTG·CAG)175 tract in pRW3712 (Fig. 2) and the
(CGG·CCG)81 tract in pRW3518 (Fig. 5) also exhibit some
instability. For the CTG·CAG repeats, the instabilities of pRW3712
are mediated by the formation of hairpins by the CTG repeats during the
synthesis of the complementary strand (Fig. 6, right). The
deletion of the CGG·CCG repeats in pRW3518 can be mediated by CGG
hairpins during the synthesis of the complementary strand. However, it
is also possible that some of these deletions are mediated by CCG
hairpins on the rolling circle template. Our experimental system does
not allow us to distinguish between deletions that occur due to CCG
hairpins from those that occur due to CGG hairpins.

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Fig. 6.
Model for in vivo
instability of TRS in a phagemid replication system.
Deletion of TRS may occur due to events during leading strand rolling
circle replication or during the synthesis of the complementary strand.
The left side shows that leading strand deletions
may be mediated by the formation of hairpin structure(s) by the TRS
tract (thick line) on the template
(minus)-strand. These hairpins are bypassed by the replication fork,
resulting in a deleted TRS tract. The right side
shows that complementary strand deletions may also occur when the
single-stranded TRS tract in the plus strand forms a hairpin structure.
This structure is bypassed by the polymerase during the synthesis of
the minus strand, resulting in a shortened TRS tract.
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In summary, since the phagemid replication system has been widely
investigated (22, 23) as a model for leading strand synthesis, our
in vivo data clearly show that the deletion of TRS can occur
on the leading strand of DNA replication. Furthermore, since the
synthesis of the complementary strand is continuous, the deletion
events observed during this step also support the leading strand
deletion model.
Different Length Hairpins Mediate TRS Deletion by Bypass DNA
Synthesis on the Rolling Circle and on the Complementary
Strand--
We observed that the direction of f1 replication has a
substantial effect on the stability of CTG·CAG and CGG·CCG repeats as evidenced by the quantitation of the extents of instabilities. The
products of the instabilities of pRW3517 and pRW3518, which contain a
(CCG·CCG)81 tract, were further characterized. For
pRW3517, the quantitation of the TRS instability is shown in Fig.
5B. The full-length (CGG·CCG)81 gave rise to
two major products containing (CGG·CCG)20 and
(CGG·CCG)6. The product containing six repeats appears
after one recultivation and increases to constitute 22% of total TRS
by the second recultivation. On the other hand, the product containing
20 repeats appears only after the third recultivation but constitutes
97% after six recultivations. Fig.
7A (left) shows that the (CGG·CCG)6 and the (CGG·CCG)20 can
arise from the (CGG·CCG)81 by the formation of long
hairpins containing ~37 and 30 CGG repeats, respectively, in the stem
of the hairpin. These deletions probably take place during the
conversion of the double-stranded RF to the single-stranded (+)-strand
by rolling circle replication. Thus, the instability of the
(CGG·CCG)81 tract in pRW3517 is mediated by long
hairpins that yield relatively few products.

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Fig. 7.
Schematic model for the formation of deletion
and expansion products of (CGG·CCG)81. The
sequential formation of the expansion and deletion products of pRW3517
and pRW3518 (Table I) was postulated by a detailed analysis of the
appearance and disappearance of the various products in Fig. 5,
B and C. A, the deletion and expansion
products of pRW3517 and the mechanism of their formation are shown. The
full-length TRS containing (CGG·CCG)81 in the
double-stranded RF may be deleted to two deletion products containing
20 repeats and six repeats by the formation of two long hairpins by the
CGG repeats (thick line) on the rolling circle
template that contain 37 and 30 duplex repeats in the stem (indicated
in the figure by the numbers next to the hairpin
loop intermediate structures), respectively. The (+)-strand that
contains (CCG)6 can undergo expansion due to the formation
of a hairpin by the CGG repeats in the newly synthesized strand during
complementary strand synthesis. B, the deletion products of
pRW3518 may be formed by a multiplicity of hairpin loop intermediates.
The deletions of pRW3518 can be mediated by the formation of hairpins
by the CGG repeats on the (+)-strand template during complementary
strand synthesis. The full-length TRS tract containing
(CGG)81 in the (+)-strand may be deleted to 52 and 39 repeats by the formation of CGG hairpins containing 14 and 20 repeat
stems, respectively. The RFs that contain the primary deletion products
yield (+)-strands (not shown). The (+)-strands containing 52 repeats
can yield an RF containing 45 repeats due to the formation of a two or
three-repeat CGG hairpin stem during complementary strand synthesis.
The (+)-strand containing 39 repeats may be deleted to 27 and 34 repeats by the formation of five- or six- and two- or three-repeat CGG
hairpin stems, respectively. The 34-repeat tract can be further deleted
to a 27-repeat-containing tract by the formation of another two- or
three-repeat CGG hairpin.
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In contrast, an analysis of the deletion products of pRW3518 (Fig.
5C) showed that the (CGG·CCG)81 was deleted to
tracts containing 52, 45, 39, 34, and 27 repeats. The full-length
(CGG·CCG)81 is gradually deleted until it constitutes
approximately 30% of total TRS after six recultivations. The loss of
the (CGG·CCG)81 coincides with the appearance of tracts
containing the (CGG·CCG)52 and the (CGG·CCG)39 by the third recultivation. Fig.
7B shows that the primary deletion products are
double-stranded RFs containing 52 and 39 repeats, which can arise due
to the formation of CGG hairpins containing ~14 and 20 repeats,
respectively, in the stems. The loss of the (CGG·CCG)52
from the third recultivation to the fourth (Fig. 5C)
corresponds to the appearance of a product containing (CGG·CCG)45, suggesting that the
(CGG·CCG)45 is a deletion product of the
(CGG·CCG)52. This secondary deletion presumably takes
place when the (CGG)52 in the single-stranded (+)-strand
synthesized from the double-stranded RF forms a small hairpin
containing three repeats in the stem (Fig. 7B). This hairpin
can be bypassed during complementary strand synthesis, resulting in an
RF containing (CGG·CCG)45.
Fig. 5C further shows that deletion products containing
(CGG·CCG)34 and (CGG·CCG)27 appear late in
the growth. Whereas the (CGG·CCG)34 constitutes 13% of
total TRS after four recultivations and then diminishes to 5% after
six recultivations, the (CGG·CCG)27 arises after four
recultivations and finally accounts for 42% after six recultivations.
The appearance of the (CGG·CCG)27 coincides with the
reduction of both the (CGG·CCG)39 and the
(CGG·CCG)34, suggesting that the
(CGG·CCG)27 is formed by the deletion of the (CGG·CCG)39 and the (CGG·CCG)34 via
different hairpin intermediates containing three or five CGG repeats in
the hairpin stem (Fig. 7B). In agreement with previous work
(10, 11), we observed that the absolute size of the deletion products
of pRW3517 and pRW3518 varied slightly from experiment to experiment,
but the overall pattern of deletions was reproducible.
Thus, the deletion of the (CGG·CCG)81 tract in pRW3518 is
mediated by a variety of CGG hairpins of different sizes on the complementary strand, which gives rise to numerous deletion products. In sharp contrast, we observe only two major deletion products from
pRW3517 as discussed earlier. For the CTG·CAG repeat, we observed
differences between the deletion products that arose from pRW3711 and
pRW3712 (Fig. 2A). The (CTG·CAG)175 tract in pRW3711 was deleted to major products containing approximately 10 and
22 repeats, presumably mediated by long CTG hairpins containing ~75-82 repeats in the stem during rolling circle replication. On the
other hand, we identified at least five major deletion products of
pRW3712 (Fig. 2A) containing
(CTG·CAG)170, (CTG·CAG)145, (CTG·CAG)135, (CTG·CAG)100, and
(CTG·CAG)32. The formation of these products is mediated
by CTG hairpins of size ranging from 2 to 70 repeats during
complementary strand synthesis.
Therefore, we propose that different length hairpin loops mediate the
deletion processes on the rolling circle template and on the
complementary strand.
(CTG·CAG)n and (CGG·CCG)n Expand during
Replication in Vivo--
In addition to deletions, we also detect
expansions of the CGG·CCG and the CTG·CAG repeats. For the
CGG·CCG repeats, an analysis of the instability products of pRW3517
(Fig. 5B) showed that a tract containing
(CGG·CCG)6 appears early in the growth, constitutes 22%
of total TRS by the second recultivation, and then disappears to less
than 1% of total TRS after six recultivations. Since a total of 76 bp
of non-TRS DNA flank the CGG·CCG repeats, it is possible to identify
deletion products that contain down to zero repeats. We found that the
disappearance of the (CGG·CCG)6 does not coincide with
the appearance of smaller deletion products. However, it does
correspond to the increase in (CGG·CCG)20, which constitutes 97% of total TRS after six recultivations. Therefore, it
is likely that a substantial portion of the (CGG·CCG)20
may have arisen due to an expansion of the (CGG·CCG)6.
The right side of Fig. 7A shows that
such an expansion may be mediated by a six- or seven-repeat CGG hairpin
formed on the newly synthesized complementary strand. In contrast, no
putative expansions were observed for the (CGG·CCG)81
tract in pRW3518 (Fig. 5, A and B). For the
CTG·CAG repeat, Fig. 2A shows that the
(CTG·CAG)175 in pRW3711 is deleted to a 10-repeat tract.
The (CTG·CGG)10 decreases to less than 1% of total TRS
by the fourth recultivation and appears to contribute to the increase
in the (CTG·CAG)20, which accounts for 95% of total TRS
after five recultivations. This suggests a possible expansion of the
(CTG·CAG)10 to a tract containing
(CTG·CAG)20 due to the formation of a CTG hairpin on the
nascent complementary strand. The length of the stem of this hairpin
intermediate could be five triplet repeats. We were unable to detect
any species that could have arisen by expansion from pRW3712 (Fig.
2A).
Thus, although deletions are the predominant instabilities observed,
expansions of the CGG·CCG and CTG·CAG repeats were also detected.
Whereas essentially all of the (CGG·CCG)6 and the
(CTG·CAG)10 were presumably expanded to larger products,
the length increases due to these putative expansions were relatively
modest. The inability to detect similar putative expansions from
pRW3712 and pRW3518 may be due to the absence of sufficiently short TRS
tracts that would be prone to expand.
Length and Orientation-dependent Instabilities of
(TTC·GAA)150--
An expansion of the
TTC·GAA repeat in the human frataxin gene results in the autosomal
recessive neurological disease Freidreich's ataxia (7). The TTC·GAA
repeats were shown (13, 61) to be unstable, depending on their
orientation relative to the unidirectional ColE1 replication origin in
pUC19 and pSPL3 vectors when propagated in various E. coli
host strains. The TTC·GAA repeats have a propensity to form
supercoil-dependent pur · pur · pyr triple helical
structures (61). However, the role of these triplexes, if any, in the
genetic instability of TTC·GAA sequences is uncertain (13, 61,
63).
In order to understand the effect of the f1 replication on these
repeats, we constructed two phagemids, pRW3545 and pRW3546 (Table I),
by cloning a tract of (TTC·GAA)150 from the human frataxin gene (13) into pGEM3Zf+ and pGEM3Zf
, respectively (see
"Experimental Procedures"). The phagemids were propagated in log
phase in E. coli NM522 for five recultivations, and the RF
DNA was digested, end-labeled, and analyzed on 5% polyacrylamide gels
(Fig. 8A). Quantitative
analyses of these gels showed that the (TTC·GAA)150
repeat in pR3546 is less stable than in pRW3545 (Fig. 8B).
Once again, it is clear that the orientation of the f1 origin has an
influence on the stability of the triplet repeat tract. Ohshima
et al. (13) showed that if the GAA repeats comprise the
template of the lagging strand when replicated from the unidirectional ColE1 origin in E. coli, the plasmid is significantly
destabilized. This is in contrast to the opposite orientation, where
TTC repeats comprise the lagging strand template. Based on these
observations, they suggested that single-stranded GAA repeats form a
more stable DNA secondary structure than the TTC strand. Our
observation that the GAA repeat in the rolling circle template results
in greater instability than the TTC repeat is consistent with this
conclusion. These deletions may be mediated by the formation of as yet
unknown secondary structures by the GAA repeats. It was proposed that deletions and expansions of the TTC·GAA repeat are mediated by the
formation of triplex structures during DNA replication (61, 63).
However, the possibility of other secondary structures being involved
in the genetic instability of these repeats cannot be ruled out.

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Fig. 8.
In vivo instability of
(TTC·GAA)150. A, pRW3545 and pRW3546
(Table I) were analyzed as described in the legend to Fig.
2A. All (TTC·GAA)150-containing restriction
fragments also contain 135 bp of nonrepetitive flanking sequence.
B, the instabilities of pRW3545 ( ) and pRW3546 ( ) were
quantitated as described in the legend to Fig. 2C from three
separate recultivation experiments. The data treatment is also as
described in the legend to Fig. 2C.
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The instability of TTC·GAA repeats in patients of Freidreich's
ataxia has been shown to be dependent on the length of the repeat tract
(7, 64). Also, the TTC·GAA repeats are unstable in plasmids in
E. coli in a length-dependent manner (13). These workers found that whereas (TTC·GAA)70 cloned in pUC19
was completely stable when propagated in E. coli SURE cells,
tracts containing (TTC·GAA)150 and
(TTC·GAA)270 were highly unstable under the same conditions. Therefore, we studied the effect of the length of the
TTC·GAA repeat on its instability in the filamentous phagemid replication system in vivo. pRW3543 and pRW3544 (Table I)
were constructed containing (TTC·GAA)70 cloned in
pGEM3Zf+ and pGEM3Zf
, respectively. Characterization of the
double-stranded RF after propagation in E. coli NM522 in the
presence of M13K07 over five recultivations revealed that both pRW3543
and pRW3544 showed the following. First, there was no difference in the
stabilities of pRW3543 and pRW3544 over five recultivations (data not
shown). Second, even after the fifth recultivation, the
(TTC·GAA)70 tract was stably maintained (<5% deleted)
(data not shown). This behavior of the shorter TTC·GAA repeat tract
is similar to that observed for the (CTG·CAG)130 tract in
pRW3111 and pRW3121 (Fig. 2). This is in agreement with the general
paradigm of length-dependent instability of TRS (1).
Interestingly, the deletion patterns of the TTC·GAA repeat do not
appear to be different for pRW3545 and pRW3546 (Fig. 8A). This is in sharp contrast to the product distribution observed for
CTG·CAG and the CGG·CCG repeats discussed earlier. The deletion products of the (TTC·GAA)150 in both pRW3545 and pRW3546
are numerous, suggesting that a variety of small hairpins or slipped
structures are responsible for this behavior.
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DISCUSSION |
Replication-based Deletion of Triplet Repeats--
Our data show
that CTG·CAG, CGG·CCG, and TTC·GAA repeats are expanded and
deleted during filamentous phage replication in vivo. It was
previously suggested (9) that the genetic instability of long tracts of
CTG·CAG sequences in duplex plasmids in E. coli is
mediated by hairpin formation on the lagging strand template during DNA
replication. We postulated (9) the lagging strand as the location of
hairpin formation by the CTG repeats based on earlier work (29) that
showed that, during DNA replication, palindromic sequences are more
likely to form secondary structures on the lagging strand template than
on the leading strand template due to misalignment. Once formed, these
secondary structures can be bypassed by the DNA replication complex,
resulting in deleted progeny strands.
The discontinuous synthesis of the Okazaki fragments on the lagging
strand involves several steps that include new primer synthesis,
polymerase cycling to the 3'-OH terminus of the new primer, synthesis
of the nascent lagging strand, and termination of Okazaki fragment
synthesis (22). Thus, from the moment the DNA is unwound by the
helicase until the completion of Okazaki fragment synthesis, the
lagging strand template is in the single-stranded state, providing an
opportunity for the formation of secondary structures.
In contrast, on the leading strand, the DNA polymerase closely follows
the DNA helicase, which unwinds the DNA. In vitro studies on
leading strand synthesis (65) showed that the rapid movement of the
replication fork depends on protein-protein interactions between the
-subunit of DNA polymerase III and the DnaB helicase. Also,
synthesis of the leading strand does not require the presence of SSB
(66). Therefore, it was proposed (22, 65) that little or no leading
strand template exists freely in the single-stranded state between the
DNA helicase and the DNA polymerase.
Model Systems for the Analysis of the Leading Strand--
Leading
strand synthesis has been studied using in vitro replication
systems in which rolling circle replication of a tailed duplex DNA
template is sustained by T4 (23) or E. coli (66, 67)
replication proteins. The advantage of using rolling circle replication
to study the leading strand lies in the ability to prevent Okazaki
fragment synthesis in these systems and thus facilitate the study of
the leading strand alone. As opposed to the replication of isometric
phages like
X174, wherein rolling circle replication occurs
concurrently with Okazaki fragment synthesis, the filamentous phages
f1, fd, and M13 synthesize the (+)-strand by rolling circle replication
and the (
)-strand by complementary strand synthesis (19). Therefore,
a phagemid replication system that uses an f1 origin is appropriate to
dissect the replication fork and investigate the continuous leading
strand synthesis in vivo.
A key difference between the f1 and the E. coli replication
systems is that these processes utilize different helicases. In E. coli, the hexameric DnaB helicase unwinds the duplex as
it tracks along the lagging strand template in the 5' to 3' direction in concert with the leading strand polymerase to which it is physically coupled (65, 68). In contrast, f1 replication utilizes the dimeric
E. coli Rep helicase, which unwinds the duplex DNA as it
tracks along the (
)-strand in the 3' to 5' direction (20, 51, 68).
Since the Rep helicase and the polymerase function independently on
(
)-strand during f1 replication (69), it is possible for substantial
single-stranded regions to be created.
Hairpin Formation on the Leading Strand Template--
We observed
frequent deletions when (CTG)175 constituted the rolling
circle template. However, the phagemid was substantially more stable
when (CAG)175 was present in the rolling circle template. In order to rule out the possibility that other factors such as plasmid-flanking sequence may have been responsible for the observed differences, we switched the orientations of the TRS in the phagemids pGEM3Zf+ and pGEM3Zf
. The experiments done with these switched phagemids confirmed our earlier conclusion that the presence of (CTG)175 in the rolling circle template was deleterious. We
also found that this behavior was dependent on the length of the CTG repeat tract, because (CTG·CAG)130 was completely stable
in this system. Since CTG repeats have been proposed to form stable
secondary structures (34), we suspected that the observed deletions
occurred due to hairpin formation in the CTG tract on the rolling
circle template. To confirm that the rolling circle template was the location for the instabilities, we studied the behavior of
(CGG·CCG)81 in this system and found that the presence of
the (CGG)81 on the rolling circle template gave
substantially more deletions than when (CCG)81 was present
in the same location. Since it was postulated (12, 48, 62) that under
in vivo conditions and at physiologically relevant lengths,
CGG was more likely than CCG to adopt stable secondary structures, we
concluded that the rolling circle template was indeed the location for
a majority of the deletions. Therefore, by extrapolation, these data
show that deletions can occur by hairpin formation on the leading
strand. Further, our observation that plasmids carrying TTC·GAA
repeats are more unstable when the GAA repeats are present in the
rolling circle template supports our contention (13) that the GAA
repeats have a higher propensity to adopt compact secondary structures
than the TTC repeats.
Based on genetic and biochemical studies on filamentous phage genome
organization, it was proposed that the minus strand was >95%
responsible for transmission of genetic information (70, 71).
Therefore, the deletion and expansion events that occur on the
(+)-strand may be more difficult to detect in the in vivo phagemid replication system. It is possible that the higher instability of TRS due to hairpin formation observed on the (
)-strand reflects this inherent bias. However, since the (+)- and the (
)-strands are
replicated by continuous DNA synthesis, the deletions and expansions
observed during both steps of phagemid replication strongly support the
leading strand deletion model.
Polymerase Pausing May Facilitate Hairpin Formation on the Leading
Strand--
We observe leading strand deletions although it was
suggested (9) that secondary structures are less likely to form on this
strand due to the absence of significant stretches of single-stranded DNA. In vivo (48) and in vitro (39, 46, 47)
experiments show that long tracts of TRS can block the progression of
DNA polymerases. In contrast, Hiasa and Marians (72) studied the in vitro bidirectional and rolling circle replication of
CTG·CAG repeats from the oriC replication origin of
E. coli and did not detect any impediment to the progression
of the replication forks through the triplet repeat tract. However,
these workers could not rule out the possibility that replication fork
stalling did occur at low frequencies that caused deletions and
expansions in vivo that were undetectable in their in
vitro assays.
Therefore, we speculate that for the in vivo f1 replication
system the TRS are able to stall the leading strand polymerase but are
unable to block the progression of the Rep helicase on the (
)-strand.
Since the polymerase and the Rep helicase are not physically coupled,
they could function independently of each other (69). Hence, any
retardation of the polymerase by the TRS would have no impact on the
progression of the helicase, and a substantial region of
single-stranded leading template is created. This single-stranded
region would have ample opportunity to adopt secondary structures that
could be bypassed by the polymerase, resulting in a deleted TRS tract.
Unlike the rolling circle template, the (+)-strand is single-stranded.
Therefore, deletions would be expected to be frequent when CTG or CGG
repeats constitute the complementary strand. However, in this case we
observe only a modest instability. This could be because SSB rapidly
binds to the tail of the (+)-strand immediately after its synthesis by
rolling circle replication and removes the secondary structures on the
(+)-strand (51), including those formed by the TRS (15). The easy
access of SSB to the secondary structures on the (+)-strand results in
their expedient removal, thus substantially obviating the deletion
process. This agrees with the observations of Rosche et al.
(15) that the SSB protein enhances the stability of CTG·CAG repeats
on plasmids in E. coli. These workers proposed that this
behavior was due to the ability of SSB to destabilize the hairpins
formed by the CTG repeats on the lagging strand during DNA replication.
Different Length Hairpins Mediate Rolling Circle and Complementary
Strand Deletions--
Our results for the CGG·CCG and the CTG·CAG
repeats show that the deletion process is different during rolling
circle replication than during complementary strand synthesis. For the
CGG·CCG repeats, when (CGG)81 was the template for
rolling circle replication, the TRS was deleted to short tracts
containing six and 20 repeats. These deletions were probably caused by
long CGG hairpins that contained 30-40 repeats in their duplex stems.
The relatively few deletion products observed indicate that these long
hairpins are quite homogenous. Alternately, the deletions are formed
early in the replication of the phagemids and eventually outgrow the longer TRS tracts (10, 11). In contrast, when the (+)-strand contains
(CGG)81, a variety of deletion products varying from 27 to
52 repeats are observed. These products arose from primary deletions of
the (CCG)81 and then secondary deletion of the primary products. We believe that the mediators of this process are hairpin structures composed of CGG repeats that vary in size from two to 20 repeats in the stem. Rolling circle replication of the
(CTG)175 template resulted in extremely few deletion
products that were 10 and 20 repeats in length, perhaps caused by the
formation of 75-80-repeat-long CTG hairpins. Replication of the
(+)-strand resulted in several deletion products that contained 32, 100, 135, 145, and 170 repeats. Therefore, we conclude that during complementary strand synthesis, deletions are mediated by a family of
hairpins with 2-20 repeats in their stems.
The differences between the sizes of the hairpins formed on the
(+)-strand and the (
)-strand can be explained by the greater probability of the SSB protein binding and destabilizing secondary structures on the (+)-strand than on a single-stranded region on the
(
)-strand. It is possible that the pausing of the DNA polymerase
during rolling circle replication and the subsequent formation of long
hairpins on the template happens during a relatively brief time frame.
Therefore, there may not be enough time for the SSB to find the
hairpins and destabilize them. On the other hand, SSB can bind the
(+)-strand as soon as it is synthesized and reeled off the rolling
circle and hence prevent the formation of long hairpins. However, at a
low frequency, small hairpins or slipped structures could be formed
that elude the SSB molecules. This would explain not only the lower
extent of deletions but also the apparently smaller hairpin
intermediates that mediate them during complementary strand synthesis.
Expansion of CTG·CAG and CGG·CCG Repeats during Complementary
Strand Synthesis--
Replication-mediated expansions of CTG·CAG and
CGG·CCG repeats were demonstrated in E. coli at a very low
frequency (9, 12, 60). These expansions were proposed to occur by the
formation of CTG and CGG hairpins on the nascent lagging strand during
DNA replication. We observed that expansion of CTG·CAG and CGG·CCG repeats can also take place during the phagemid replication cycle and
occur at a high frequency. For the CTG·CAG repeat, we detected the
appearance of a (CTG·CAG)20 that coincided with the loss
of a (CTG·CAG)10 when CTG repeats were in the newly
synthesized (
)-strand. Also, an apparent expansion of a
(CGG·CCG)6 to a (CGG·CCG)20 was observed
when CGG was in the newly synthesized (
)-strand. Overall, these data
suggest that the putative expansions were mediated by a five-repeat CTG
hairpin and a six- or seven-repeat CGG hairpin formed on the
nascent (
)-strand during complementary strand synthesis.
We do not observe expansions when the CTG and CGG repeats are present
on the (+)-strand. This may be indicative of the higher probability of
expansions during complementary strand synthesis. However, the only
putative expansions observed arose secondarily from extremely short
deletion products of the full-length TRS. Since the highly deleted TRS
tracts were observed only in one orientation, we cannot rule out the
possibility that expansions could occur during rolling circle
synthesis, provided that the TRS was deleted to an optimal length. At
this length, the TRS could be more prone to expansion than deletion.
TTC·GAA Repeat Instability Differs from the Other Triplet
Repeats--
Interestingly, the behavior of the TTC·GAA repeats is
quite different from that of the CTG·CAG and CGG·CCG repeats.
Whereas the CTG·CAG and the CGG·CCG repeats delete via different
hairpin intermediates for the two orientations of the f1 origin, the
TTC·GAA repeats show no such differences. A variety of deletion
products of (TTC·GAA)150 were observed whether TTC or GAA
was present on the rolling circle template. These deletion products
covered almost the entire range of repeat lengths between 150 and 20 repeats. Thus, we propose that these deletions were mediated by an
assortment of TTC or GAA secondary structures on both the (+)-strand
and the (
)-strand, presumably by small slippage events.