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J. Biol. Chem., Vol. 275, Issue 40, 31528-31535, October 6, 2000
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From the Department of Biological Sciences, University of Alberta,
Edmonton, Alberta T6G 2E9, Canada
Received for publication, May 26, 2000, and in revised form, July 27, 2000
DNA replication normally occurs with high
fidelity, but certain "slippery" regions of DNA with tracts of
mono-, di-, and trinucleotide repeats are frequently mutation hot
spots. We have developed an in vitro assay to study the
mechanism of dinucleotide repeat expansion. The primer-template
resembles a base excision repair substrate with a single nucleotide gap
centered opposite a tract of nine CA repeats; nonrepeat sequences flank
the dinucleotide repeats. DNA polymerases are expected to repair the
gap, but further extension is possible if the DNA polymerase can
displace the downstream oligonucleotide. We report here that the wild
type bacteriophage T4 DNA polymerase carries out gap and strand
displacement replication and also catalyzes a dinucleotide expansion
reaction. Repeat expansion was not detected for an
exonuclease-deficient T4 DNA polymerase or for Escherichia
coli DNA polymerase I. The dinucleotide repeat expansion reaction
catalyzed by wild type T4 DNA polymerase required a downstream
oligonucleotide to "stall" replication and 3' DNA polymerases replicate DNA with high fidelity except for
certain DNA sequences, the so-called "slippery" DNAs, which are tracts of simple repeat sequences (reviewed in Ref. 1). The lengths of
tracts of mono-, di-, and trinucleotide repeats are unstable, which can
easily be detected as repeat-length polymorphisms in microsatellite
sequences or as mutation hot spots, such as the classical frameshift
hot spots identified by Benzer in the bacteriophage T4 rII
genes (2), which are tracts of six A nucleotides (3).
Hypermutability in repeat tracts can have serious consequences for
human health as observed for an inherited mutation in the human
APC gene, which converts the wild type sequence AAATAAAA to
the A8 mononucleotide tract (4). The A8
sequence was found to create a small hypermutable region for gene
inactivating mutations that predispose carriers to colorectal cancer
(4). Streisinger et al. (5) suggested that frameshifts are
produced in repeat sequences by a transient separation of the primer
and template strands and then misalignment during reannealing to
generate an intermediate in which one or more repeats is unpaired. This
"slippage" intermediate is usually repaired by postreplication
mismatch repair as revealed by the dramatic increase in repeat
instability when this repair pathway is inactivated (6-8).
In vitro assays have been used to measure DNA
polymerase-catalyzed "reiterative replication" of repeat sequences
(for examples, see Refs. 9-14). The number of repeats can be amplified
several hundred-fold by a variety of DNA polymerases, but most of the synthetic DNA substrates used are composed exclusively of repeat sequences. One objective of our studies was to develop an improved in vitro assay utilizing a DNA substrate that more closely
resembles genomic DNA in which the repeat tract is flanked by nonrepeat sequences. A second objective was to use the more natural DNA substrate
to probe the mechanism of DNA polymerase slippage. Both objectives were
achieved with a DNA substrate in which a single nucleotide gap was
positioned opposite the center of the dinucleotide repeat sequence
(AC)9. The AC dinucleotide repeat was chosen because poly(AC/GT)10-30 is the most common simple repeat in many eukaryotic genomes (15). Nonrepeat sequences were positioned on both
sides of the (AC)9 dinucleotide tract, as found in genomic DNA. The nonrepeat sequences also allowed precise annealing of the
template and primer strands. A single nucleotide-gapped substrate is
produced in vivo during base excision repair. A similar
substrate is also produced when DNA polymerases have nearly completed
repair synthesis of the larger gaps formed by nucleotide excision
repair and postreplication mismatch repair and during lagging strand replication when the replicating DNA polymerase reaches the RNA primer
of the next Okazaki fragment.
We examined replication of the single nucleotide-gapped DNA substrate
by wild type and exonuclease-deficient T4 DNA polymerases and by
Escherichia coli DNA polymerase I. Dinucleotide expansion was detected only for the wild type T4 DNA polymerase. While many previous in vitro assays detected repeat expansion by
exonuclease-deficient DNA polymerases, our studies demonstrate that not
only does the wild type T4 DNA polymerase, which has a potent 3' Materials
DNA Polymerases--
Purifications of the bacteriophage T4 wild
type and the exonuclease-deficient D112A/E114A-DNA polymerases have
been described (16, 17). E. coli DNA polymerase I and Klenow
fragment were purchased from Amersham Pharmacia Biotech.
DNA Substrates--
The DNA substrates used in this study are
described in Fig. 1. The oligonucleotides were synthesized using
standard procedures by the DNA synthesis facility in the Department of
Biological Sciences at the University of Alberta. Two types of
hydrolysis-resistant oligonucleotides were prepared. A 3'-phosphate was
introduced by using a phosphate-derivatized CPG (Glen Research,
Sterling, VA). A 3'-phosphate prevents both DNA polymerase nucleotide
incorporation and 3'
The template and primer strands were annealed in buffer containing 50 mM Tris-HCl, pH 8.0, and 25 mM NaCl. The
32P-labeled oligonucleotide was present at 50 nM, and unlabeled oligonucleotides were present in 2-fold
excess at 100 nM. The solution was heated at 80 °C for 5 min and slow cooled to room temperature (90 min).
Methods
Reaction Conditions--
Reaction mixtures (50 µl) contained 5 nM DNA substrate, 50 nM DNA polymerase, 67.5 mM Tris-HCl, pH 7.5, 25 mM NaCl, 100 µM dNTPs, 0.5 mM dithiothreitol, 0.2 mg/ml
bovine serum albumin, and 0.5 mM EDTA. The mixtures were
preincubated at 37 °C for 5 min, and then reactions were started by
the addition of MgCl2 (final concentration, 8 mM). Reactions were stopped at various times by
removing 5-µl samples and mixing with an equal volume of gel loading
buffer, which contained formamide and gel buffer. Denaturing gel
electrophoresis in 15% polyacrylamide plus 8 M urea
was used to separate the reaction products. The 32P-labeled
products were visualized by using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Chemical Sequencing--
32P-Labeled reaction
products were cut from preparative polyacrylamide gels and purified as
described above. The Maxam and Gilbert sequencing procedure was
followed (20).
Primer Extension--
In vitro replication of the AC
repeat-containing template strand was first measured in a primer
extension assay. The 32P-labeled primer was annealed to the
template strand (Fig. 1a). T4
DNA polymerase extended the primer to produce full-length 30-mer product within the first 15 s of reaction (Fig.
2A). No products longer than
the 30-mer were detected.
Dinucleotide Expansion at a Gap--
A single nucleotide gap was
created by annealing the 32P-labeled primer plus a second,
"downstream" oligonucleotide that is complementary to the 5'-end of
the template strand (Fig. 1b). A time course for replication
of the gapped DNA substrate by T4 DNA polymerase is shown in Fig.
2B. After 15 s of replication, the primer was extended
by one nucleotide to fill in the gap (14-mer), but longer 15-mer,
16-mer, and 18-mer products as well as high amounts of full-length
30-mer product were also produced. Synthesis of products longer than 14 nucleotides indicates that the T4 DNA polymerase can displace the
downstream oligonucleotide. At later time points, the amounts of the
shorter 14-, 15-, and 16-mer products decreased, while replication
products longer than the full-length 30-mer were produced. By 1 min,
and more clearly at 10 min, products were synthesized that appeared to
be longer than 30 nucleotides by the addition of dinucleotide units,
the gel bands indicated as +2, +4, +6, etc. At 10 min, repeat expansion
products represented about 13% of the replication products that were
full-length or longer.
The band indicated at the +2-position (Fig. 2B) was excised
from a preparative gel and sequenced by the Maxam and Gilbert chemical
sequencing method (20); the sequencing gel is shown in Fig.
3. The primary sequence for the +2
product has 10 GT repeats instead of the nine expected from the nine AC
repeats in the template strand. The 10 GT repeats are followed by the
nonrepeat ACCCCACC sequence. A faint secondary sequence with an
additional GT repeat can also be seen in the region of the bracket
(Fig. 3), which is due to the presence of a second minor DNA product.
For the longer apparent +4, +6 products, etc., there are 11, 12, etc. GT repeats, respectively, which are followed by the nonrepeat sequence
(data not shown). Thus, wild type T4 DNA polymerase catalyzes a
dinucleotide expansion reaction, which is followed by accurate replication of the nonrepeat template sequence.
The experiments were repeated with a similar DNA substrate but with 20 AC repeats instead of nine in the template strand. The same primer and
downstream oligonucleotides were used, which produced a single-stranded
gap of 23 nucleotides. Dinucleotide expansion by the wild type T4 DNA
polymerase was detected with the longer DNA substrate to the same
extent as observed with the shorter DNA substrate (data not shown).
Repeat Expansion by the Exonuclease-deficient D112A/E114A-T4 DNA
Polymerase--
Experiments reported in Fig. 2 with the wild type T4
DNA polymerase were repeated with the exonuclease-deficient
D112A/E114A-DNA polymerase. No dinucleotide repeat expansion was
detected for the exonuclease-deficient T4 DNA polymerase with either
the primer extension (Fig. 4A)
or the single nucleotide-gapped DNA substrate (Fig. 4B).
Displacement synthesis was more rapid by the exonuclease-deficient T4
DNA polymerase compared with the wild type enzyme, since little pausing
to produce the gap-filled 14-mer product or other products shorter than
full-length was observed (Fig. 4B, 15 s). A product apparently one nucleotide longer than the full-length product (+1) was
detected at 10 min, which is probably due to the nontemplated extension
of the primer strand that is observed for many exonuclease-deficient DNA polymerases at the ends of duplex DNA. Since dinucleotide expansion
was not detected for the exonuclease-deficient T4 DNA polymerase,
expansion by the wild type enzyme must require 3'
Dinucleotide expansion was detected for the exonuclease-deficient T4
DNA polymerase, however, if only two of the four dNTPs, dGTP and dTTP,
were supplied (Fig. 5B), but
not for the wild type T4 DNA polymerase (Fig. 5A).
Replication terminated for the wild type DNA polymerase once
replication of the repeat region was completed (22-mer). For the
exonuclease-deficient T4 DNA polymerase, a major product with an extra
GT repeat (+2) was detected within the first minute of reaction. With
longer reaction times, the amount of +2 product decreased, while the
amount of +4 and longer products increased. Thus, elongation of
misaligned primer-termini by the exonuclease-deficient T4 DNA
polymerase produces relatively stable replication products. The absence
of dinucleotide expansion products (longer than the 22-mer) for the
wild type T4 DNA polymerase indicates that exonucleolytic proofreading
efficiently corrects any expansion products. The presence or absence of
the downstream oligonucleotide had no effect on reactions with dGTP and
dTTP.
Mechanism of Dinucleotide Expansion by the Wild Type T4 DNA
Polymerase--
In order to determine at what step exonuclease
activity is needed for the dinucleotide expansion catalyzed by the wild
type T4 DNA polymerase, the three 3'-ends present in the gapped DNA substrate were modified. Besides the primer terminus, the downstream oligonucleotide and the template strand have 3'-ends that are potential
substrates for the T4 DNA polymerase 3'
Dinucleotide expansion was reduced to an almost undetectable level by
phosphorylation of the 3'-end of the template strand (Fig. 1,
d and e; Fig. 6, lanes 2 and
3, respectively). These results demonstrate that exonuclease
trimming at the 3'-end of the template strand is required for
production of high amounts of expansion products. The extent of
exonuclease digestion of the template strand required to support
dinucleotide expansion was determined by placing nonbridging
phosphorothioates at various positions at the 3'-end of the template
strand. Dinucleotide expansion was restored only if the
phosphorothioate was placed so that T4 DNA polymerase 3'
Removal of the nonrepeat sequence, however, still did not allow the
exonuclease-deficient T4 DNA polymerase to carry out the expansion
reaction. Since the exonuclease-deficient T4 DNA polymerase may extend
the phosphorothioate-modified DNA and resynthesize the nonrepeat
sequence, the experiment was repeated with a DNA substrate in which the
3'-end of the template strand was protected with a 3'-phosphate, which
prevents both degradation and extension (Fig. 1g). Products
longer than full length were detected for the wild type (Fig.
7A) and 3'
The exonuclease trimming of the template strand suggested to us that
DNA replication may also be taking place at the 3'-end of the template
strand. This proposal was tested by using the same DNA substrate that
supports dinucleotide expansion of the primer (Fig. 1c),
except that the 5'-end of the template strand was labeled with
32P (Fig. 1h). Dinucleotide expansion in the
template strand was detected only if the downstream oligonucleotide was
present (Fig. 8), as observed for
expansion in the primer strand (Fig. 2B). The apparent +2
template product was sequenced (Fig.
9). An extra dinucleotide repeat (AC) was
present, and the repeat sequence was followed by the adjacent nonrepeat
sequence.
Repeat Expansion by E. coli DNA Polymerase I--
The experiments
with the primer extension and gapped DNA substrates were repeated with
E. coli DNA polymerase I and Klenow fragment. No
dinucleotide expansion was detected (data not shown). Rapid
displacement replication was observed for both bacterial DNA
polymerases, as observed for the exonuclease-deficient T4 DNA
polymerase (Fig. 4).
We have demonstrated that the wild type T4 DNA polymerase, which
has a potent 3' The first step in the repeat expansion reaction is DNA polymerase
pausing and dissociation during replication of a repeat tract, which
was forced in our assay due to the presence of a downstream
oligonucleotide (Fig. 10A).
Although our experiments were with the T4 DNA polymerase and not the
highly processive T4 DNA polymerase holoenzyme, rapid dissociation for
the holoenzyme is also observed when replication is barred under
conditions that mimic an encounter with the 5'-end of an Okazaki
fragment (21). Thus, results presented in this report are consistent
with the proposal by others that frameshift mutagenesis is stimulated
by DNA polymerase dissociation and reassociation (13, 22). We have
extended these findings by demonstrating that DNA polymerase encounters
with downstream DNA stimulate repeat expansion (Fig. 2, compare
A and B). Furthermore, dramatic expansion may
result if repeat expansion can take place on both strands (Figs. 6 and 8). These results suggest that DNA damage resulting in strand breaks on
both strands within a repeat tract, as illustrated in Fig.
10A, has the potential, if acted on by DNA polymerases, to greatly expand the length of the repeat tract.
The second step is continued primer extension with displacement of the
downstream strand. This step may involve several cycles of DNA
polymerase dissociation and reassociation. Displacement of the 5'-end
of the downstream strand creates a "flap" of unpaired single-stranded DNA (Fig. 10A). The 5'-flap structure may be
in equilibrium with a structure in which the 3'-end of the primer strand is unpaired (3'-flap). The 3'-flap structure may spontaneously convert to a structure with a base-paired primer terminus but with one
or more internal repeats not base-paired. Alternatively, the DNA
polymerase may facilitate strand misalignment by binding the 3'-end of
the single-stranded flap directly in the exonuclease active center (23)
but then occasionally transferring the primer end to the polymerase
active center in a misaligned configuration (24,
25).1 In either case,
extension of the misaligned primer strand results in expansion of the
repeat tract.
The proposed role of a 5'-flap structure in repeat expansion is
supported by the finding that deletion of the RAD27 gene in Saccharomyces cerevisiae increases repeat length
instability, primarily to expand repeat tracts (26-28). The
RAD27 gene encodes a flap endonuclease. If this activity is
missing, then persistent flap structures may increase the opportunity
to produce the misaligned DNA structures illustrated in Fig.
10A.
Several points in this model warrant further discussion. First, there
was greater dinucleotide expansion activity with the in
vitro assay than would be expected in vivo.
Dinucleotide expansion may be limited in vivo if the optimal
conditions for expansion are not met. For example, strand
breakage in both strands within or near a repeat tract (Fig.
10A) is probably a rare event. Rapid DNA ligation or binding
of other proteins to strand discontinuities would prevent DNA
polymerase binding. The cell may also selectively utilize DNA
polymerases for gap repair that are less prone to frameshift infidelity
than T4 DNA polymerase, such as E. coli DNA polymerase I,
which does not catalyze detectable repeat expansion in our in
vitro reaction. The 5' Another point to explain is the low level of dinucleotide expansion by
the exonuclease-deficient T4 DNA polymerase in our in vitro
assays (Figs. 4 and 7) but the high amount of repeat length instability
detected for exonuclease-deficient DNA polymerases in vivo,
such as for the exonuclease-deficient yeast DNA polymerase Exonuclease-deficient DNA polymerases may increase misalignment
mutagenesis in vivo because of increased ability to displace downstream DNA. The exonuclease-deficient T4 DNA polymerase is more
proficient at displacement replication than the wild type T4 DNA
polymerase (compare Figs. 2 and 4, 15-s time points) because the
elongating primer strand is not subject to proofreading by the mutant.
A synergism between DNA polymerase proofreading and removal of RNA
primers and 5'-flap structures has been observed in yeast; a haploid
strain with an exonuclease-deficient DNA polymerase Exonuclease-deficient DNA polymerases may also be more prone to
misalignment errors at sites of DNA damage than exonuclease-proficient DNA polymerases. DNA damage presents a severe block to replication (Fig. 10B). Exonuclease-deficient DNA polymerases may
synthesize extra copies of the repeat as was observed in the reaction
with dGTP and dTTP (Fig. 5B) and as illustrated in Fig.
10B. The wild type T4 DNA polymerase did not catalyze
dinucleotide expansion when just dGTP and dTTP were supplied (Fig.
5A) because of correction by exonucleolytic proofreading;
hence, less repeat expansion at DNA damage sites is expected for
proofreading-proficient DNA polymerases.
In conclusion, we have identified conditions in an in vitro
assay that stimulate dinucleotide expansion by a wild type DNA polymerase that functions in chromosome replication. The primary instigator of DNA polymerase-catalyzed expansion is that DNA
replication is hindered within a repeat tract. The assay provides a
starting point to further probe the mechanism(s) of DNA
polymerase-catalyzed repeat expansion.
We thank Tom Petes and Christopher Pearson
for helpful comments on the manuscript and Randy Nonay and Adrian
Dobrowsky for preliminary studies.
*
This work was supported in part by Medical Research Council
of Canada Grant MT-13651 (to L. R-K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Scientist of the Alberta Heritage Foundation for Medical Research.
To whom correspondence should be addressed. Tel.: 780-492-5383; Fax:
780-492-9234; E-mail: LREHA@gpu.srv.ualberta.ca.
Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M004594200
1
M. I. Hadjimarcou, R. Kokoska, T. D. Petes, and L. J. Reha-Krantz, manuscript in preparation.
2
L. J. Reha-Krantz, unpublished data.
Dinucleotide Repeat Expansion Catalyzed by Bacteriophage T4 DNA
Polymerase in Vitro*
and
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease
activity to remove the 3'-nonrepeat sequence adjacent to the repeat
tract in the template strand. These results suggest that dinucleotide
repeat expansion may be stimulated in vivo during DNA
repair or during processing of Okazaki fragments.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'
exonuclease activity, catalyze repeat expansion, but that the
exonuclease activity stimulates the expansion reaction. We have
proposed a model for repeat expansion based on our new findings.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease activities. Hydrolysis activity
was prevented specifically at the 3'-end of the primer strand by a
nonbridging phosphorothioate (Sp isomer). The
phosphorothioate modification inhibits 3'5' exonuclease activity,
but the phosphorothioate-modified primer still supports the nucleotide
incorporation reaction. Phosphorothioate oligonucleotides were prepared
using a sulferizing reagent (Glen Research). Two diastereomers,
Rp and Sp, are produced in about equal amounts,
but the Rp-phosphorothioate oligonucleotide is degraded by
the 3'5' exonuclease activity of T4 DNA polymerase while leaving the
exonuclease-resistant Sp-oligonucleotide (18, 19). All
oligonucleotides were purified by gel electrophoresis. The appropriate
bands were cut from the gels, and the DNA was eluted from the gel
slices and further purified by chromatography through Sep-Pak
cartridges (Waters). The primer or template strands were labeled at the
5'-end by T4 polynucleotide kinase and [
-32P]ATP.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA substrates constructed to detect
dinucleotide expansion. Nine CA repeats are present in the
template strand and are underlined; the CA repeats are
flanked by nonrepeat sequences. What we define as the primer strand in
this report is labeled with 32P at the 5'-end, except for
in Fig. 1h. The GT repeat sequences in the primer strand and
in the downstream oligonucleotide are underlined.
s indicates the location of the nonbridging
phosphorothioate, Sp isomer. p indicates a
phosphate group at the indicated 3'-position. In the sketches of the
DNA substrates, repeat sequences are illustrated as open
rectangles, and the nonrepeat sequences are illustrated as
solid lines. An asterisk indicates the
position of the 32P label.
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Fig. 2.
Primer extension and dinucleotide expansion
by the wild type T4 DNA polymerase. Primer extension
(A) and gap repair assays (B) were done with the
DNA substrates described in Fig. 1, a and b,
respectively. The reaction conditions are described in detail under
"Experimental Procedures." The DNA replication products were
separated by electrophoresis on a denaturing, 15% polyacrylamide gel.
Samples were analyzed after 15 s, 30 s, 1 min, and 10 min of
reaction. Lane C contains labeled primer and
full-length DNAs. In B, the 14-mer product, which fills the
gap, and longer 15-mer, 16-mer, 18-mer, and 20-mer products that were
produced during displacement of the downstream oligonucleotide are
indicated. Apparent dinucleotide expansion products are indicated as
+2, +4, +6, etc.
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Fig. 3.
DNA sequence of the +2 dinucleotide expansion
product. Products of chemical reactions to detect modified G,
modified T, modified A+G, and modified C by the Maxam and Gilbert
procedure (20) are in the indicated lanes. The DNA sequence is given by
each band. There are 10 GT dinucleotide repeats followed by the
nonrepeat ACCCCACC sequence. The bracket indicates bands due to a minor
contaminate, which has an additional GT repeat.
5' exonuclease
activity at some step in the expansion reaction.
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Fig. 4.
Primer extension by an exonuclease-deficient
T4 DNA polymerase. The same DNA substrates used in Fig. 2 to
assess replication by the wild type T4 DNA polymerase were used for the
exonuclease-deficient D112A/E114A-DNA polymerase. Panel A,
the primer-extension assay; Panel B, gap repair and strand
displacement replication. Samples were analyzed after 15 s,
30 s, 1 min and 10 min of reaction. Lane "C" contains labeled
primer and full-length DNAs. No dinucleotide expansion was detected,
but an apparent nontemplated extension was observed at the +1 position
after 10 min of reaction.
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Fig. 5.
Primer extension in the presence of dGTP and
dTTP. The DNA substrate is described in Fig. 1a, except
that a phosphate was added to the 3'-end of the template strand to
prevent degradation of the template by the 3' 5' exonuclease activity
of the wild type T4 DNA polymerase. The same reaction conditions were
used as for Figs. 2 and 4 except that only two nucleotides, dGTP and
dTTP at 100 µM, were supplied. Panel A, primer
extension by the wild type T4 DNA polymerase. Panel B,
primer extension by the exonuclease-deficient D112A/E114A-DNA
polymerase. Primer extension in the presence of dGTP and dTTP could
continue for 9 nucleotides to produce the 22-mer. Longer products were
detected for the exonuclease-deficient T4 DNA polymerase. Apparent
dinucleotide repeat expansion products are indicated at the +2 and +4
positions.
5' exonuclease activity. A
nonbridging phosphorothioate (Sp isomer) was placed at the
3'-terminal phosphodiester bond in the primer strand to protect the
primer from exonuclease digestion, while still allowing the primer to
be used for nucleotide incorporation. Dinucleotide expansion was
detected with the phosphorothioate-modified primer (Fig.
6, lane 1) and when
the downstream oligonucleotide was also modified with a 3'-phosphate,
which prevents both exonuclease and nucleotide incorporation reactions
(Fig. 1c; Fig. 6, lane 4). Protection of the
3'-ends of the primer and downstream oligonucleotide from exonuclease
digestion increased the expansion reaction. About 21% of the
full-length and longer replication products in Fig. 6, lane
1, were expansion products and 56% in Fig. 6, lane 4, but only 13% of the replication products were expansion products in
the absence of any 3'-protection after a 10-min reaction (Fig. 2B).
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Fig. 6.
The effect of DNA modifications on the
dinucleotide expansion reaction. Reactions (10 min) catalyzed by
the wild type T4 DNA polymerase with various DNA substrates are shown.
The standard reaction conditions were used. Lane 1,
dinucleotide expansion with a single nucleotide-gapped substrate like
Fig. 1c, except that the downstream oligonucleotide was not
phosphorylated at the 3'-end. Lanes 2 and 3,
reactions with gapped DNA substrates, in which the template strands
were phosphorylated on the 3'-ends (Fig. 1d and
e, respectively). Lane 4, dinucleotide expansion
with the gapped DNA substrate described in Fig. 1c, which
has an Sp phosphorothioate at the primer-terminus and the
downstream oligonucleotide is phosphorylated at the 3'-end. Lane
5, the template strand was synthesized without the nonrepeat
sequence at the 3'-end; the 3'-end was protected with an Sp
phosphorothioate (Fig. 1f). Lane C, labeled
primer and full-length DNAs.
5'
exonuclease activity could remove the 3'-terminal nonrepeat sequence in
the template strand. This point was demonstrated by constructing a
template strand without the nonrepeat sequence and with an
Sp phosphorothioate at the 3'-end to prevent further exonuclease trimming (Fig. 1f); a strong dinucleotide
expansion reaction (83% expansion product compared with 17%
full-length) was observed (Fig. 6, lane 5). Also note that
without the 3'-terminal nonrepeat sequence in the template strand,
there was some imprecise annealing of the primer strand, which produced
a few products shorter than the full-length 30-mer.
5'
exonuclease-deficient DNA polymerases (Fig. 7B). The
distinctive dinucleotide expansion ladder was detected for the wild
type T4 DNA polymerase but not for the exonuclease-deficient DNA
polymerase; instead, products longer than the full-length template
strand increased in length by apparent single nucleotide extensions,
+1, +2, +3, etc. The single nucleotide extensions were probably
produced by a combination of dinucleotide expansion followed by an
untemplated primer extension, which is catalyzed by
exonuclease-deficient DNA polymerases (Fig. 4). Products shorter than
the full-length 30-mer were also observed (indicated by
asterisks). These products appear to be due to imprecise
primer annealings in which there may be base pairing between the G
residues in the nonrepeat sequence of the primer strand and the C
residues in the repeat tract in the template strand.
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Fig. 7.
Dinucleotide expansion with a DNA substrate
in which the 3'-end of the template strand terminates with the repeat
sequence. The DNA substrate is described in Fig. 1g.
Standard reaction conditions with all four dNTPs were carried out with
the wild type T4 DNA polymerase (panel A) and with the
exonuclease-deficient D112A/E114A-DNA polymerase (panel B).
Reaction samples were taken at the indicated times and the products
were separated by denaturing gel electrophoresis. Arrows indicate the
positions of the 13-mer primer and the full-length 30-mer. The
dinucleotide expansion ladder is observed for the wild type T4 DNA
polymerase (panel A) and products longer than full-length
are also observed for the exonuclease-deficient T4 DNA polymerase
(panel B). The asterisks indicate shorter DNA replication
products produced by imprecise annealing of the primer strand.
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Fig. 8.
Dinucleotide expansion in the template
strand. The reaction conditions were the same as used to measure
dinucleotide expansion in the primer strand. The single
nucleotide-gapped DNA substrate is described in Fig. 1h.
Reaction samples were analyzed after 1 min, 2 min, 5 min, and 10 min of
reaction. Products longer than the full-length 30-mer are indicated as
+2, +4, +6 etc.
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Fig. 9.
DNA sequence of the template +2 dinucleotide
expansion product. Products of chemical reactions to detect
modified G, modified T, modified A+G, and modified C by the Maxam and
Gilbert procedure (20) are in the indicated lanes. The DNA sequence is
given by each band. There are 10 AC dinucleotides that are flanked by
the nonrepeat sequences.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease activity (16), can catalyze a
dinucleotide expansion reaction with a single nucleotide-gapped DNA
substrate with nine AC repeats in the template strand in the presence
of all four dNTPs (Fig. 2B). Since the dinucleotide repeats are embedded between nonrepeat sequences, this assay allows us to study
dinucleotide expansion under conditions that may occur naturally in the
cell during repair of gaps produced by various DNA repair activities
and when lagging strand replication reaches the primer for the next
Okazaki fragment. The expansion products have extra dinucleotide
repeats that are followed by the adjacent nonrepeat sequence (Fig. 3).
Thus, this assay detects both the strand misalignment phase of the
expansion reaction and the switch to accurate replication of the
nonrepeat template sequence. The critical step for repeat expansion was
the requirement that DNA replication be hindered within the repeat
tract. We combined results from in vitro dinucleotide
expansion reactions with in vivo observations to formulate
the following models for repeat expansion.
View larger version (9K):
[in a new window]
Fig. 10.
Model for repeat expansion catalyzed by the
T4 DNA polymerase. Panel A, misalignment mutagenesis
promoted by displacement replication. Panel B, misalignment
mutagenesis promoted by DNA damage (X) in the template strand. The
thicker lines represent repeat tracts. The black thick lines represent
the primer strands and potential sites for strand misalignment.
3' exonuclease activity of E. coli DNA polymerase I would prevent formation of a 5'-flap and, thus, reduce opportunity for strand misalignment.
(29) and
for the T4 D112A/E114A-DNA
polymerase.2 There are two
reasons why exonuclease deficiency reduces repeat expansion in our
assay. First, there is little expansion detected if the nonrepeat
sequence at the 3'-end of the template strand cannot be removed by
exonuclease degradation (Fig. 6, lanes 2 and 3).
Second, the short downstream oligonucleotide was displaced and
dislodged too readily by the exonuclease-deficient T4 DNA polymerase,
which means that primer elongation within the repeat tract was not
hindered as much for the exonuclease-deficient DNA polymerase as for
the wild type enzyme. When primer elongation was confined to the repeat
tract, however, as forced in reactions in which only dGTP and dTTP were
supplied, the exonuclease-deficient T4 DNA polymerase catalyzed a
strong expansion reaction (Fig. 5B).
and no flap
endonuclease is not viable (28). The lethal phenotype of the double
mutant may be due to the higher production of 5'-flap structures due to
increased strand displacement replication by the exonuclease-deficient
DNA polymerase compared with the wild type enzyme, which could lead
to persistent strand discontinuities and/or intolerable levels of
misalignment mutagenesis (error catastrophe).
ACKNOWLEDGEMENTS
FOOTNOTES
Supported, in part, by CAPES (Brasília-Brazil) and the
Universidade Gama Filho, Rio de Janeiro, Brazil.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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