Research Article
3867
DNA replication and transcription direct a DNA strand
bias in the process of targeted gene repair in
mammalian cells
Erin E. Brachman and Eric B. Kmiec*
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
*Author for correspondence (e-mail: [email protected])
Accepted 6 April 2004
Journal of Cell Science 117, 3867-3874 Published by The Company of Biologists 2004
doi:10.1242/jcs.01250
Summary
The repair of point mutations can be directed by modified
single-stranded DNA oligonucleotides and regulated by
cellular activities including homologous recombination,
mismatch repair and transcription. Now, we report that
DNA replication modulates the gene repair process by
influencing the frequency with which either DNA strand is
corrected. An SV40-virus-based system was used to
investigate the role of DNA synthesis on gene repair in
COS-1 cells. We confirm that transcription exerts a strand
bias on the gene repair process even when correction takes
place on actively replicating templates. We were able to
distinguish between the influences of transcription and
Key words: DNA repair, SV40, COS cells
Introduction
Targeted nucleotide exchange, the underlying mechanism of
gene repair, can direct the alteration of single bases in
biological systems including cell-free extracts, Escherichia
coli, Saccharomyces cerevisiae, mammalian cells in culture
and a range of animal models including mouse, rat and
dog (Alexeev et al., 2000; Alexeev and Yoon, 1998;
Bandyopadhyay et al., 1999; Bartlett et al., 2000; Brachman
and Kmiec, 2002; Brachman and Kmiec, 2003; Campbell et
al., 1989; Cole-Strauss et al., 1996; Cole-Strauss et al., 1999;
Ellis et al., 2001; Gamper et al., 2000; Kmiec, 1999; Kren et
al., 1997; Kren et al., 1999; Liu et al., 2001; Moerschell et al.,
1988; Parekh-Olmedo et al., 2001; Rando et al., 2000; Rice et
al., 2001; Santana et al., 1998; Vasquez et al., 2001; Yamamoto
et al., 1992a; Yamamoto et al., 1992b; Yoon et al., 1996). Now,
efforts are under way to elucidate the mechanism of this
dynamic process so that correction efficiencies can be
stabilized and eventually raised to therapeutically useful levels.
Genetic studies in yeast have revealed that DNA repair proteins
play regulatory roles in oligonucleotide-directed repair
(Brachman and Kmiec, 2003; Liu et al., 2002a). Additionally,
transcriptional activity of the target gene has been found to
provide a more open chromatin conformation, which enables
a higher frequency of gene repair (Igoucheva et al., 2003; Liu
et al., 2002b). A strand bias has repeatedly been observed in
which the untranscribed strand is corrected more efficiently
(Igoucheva et al., 2001; Igoucheva et al., 2003; Liu et al., 2001;
Liu et al., 2002b; Nickerson and Colledge, 2003) (Y. Hu, L.
Liu, L. Ferrara and E.B.K., unpublished) and lends further
support for a productive role of transcription in the gene repair
process. This transcriptional strand bias is probably due to the
availability of the untranscribed strand for oligonucleotide
alignment, in contrast to the transcribed strand, which is more
encumbered by the transcriptional machinery. Furthermore,
experiments conducted in vitro indicate that RNA polymerase
is able to displace the oligonucleotide from the transcribed
strand, reducing its potential for directing a gene repair event
(Liu et al., 2002b).
Transcriptional activity alone, however, does not always
dictate the strand bias of correction. For example, when
targeted nucleotide exchange takes place on the S. cerevisiae
CYC1 gene, a gene involved in cytochrome c metabolism, a
repair bias is observed that favours the transcribed strand
(Brachman and Kmiec, 2003; Yamamoto et al., 1992b). Similar
results have now been reported in two unrelated mammalian
systems (Bertoni et al., 2003; Bertoni and Rando, 2003;
Sorensen et al., 2003), suggesting that processes other than
transcription can impact strand bias. In the current work, we
examine the possibility that DNA replication influences the
process of gene repair strand preference.
Support for this notion comes from important studies in
prokaryotes in which DNA replication has already been shown
to dominate the strand bias phenomenon (Ellis et al., 2001;
Zhang et al., 2003). Recombination within the gal operon of
E. coli is higher when oligonucleotides complementary to the
lagging strand of replication are used. galK targeting revealed
that the bias for the lagging strand remained intact even when
the gal operon was inverted, reversing the strands of replication
replication on strand bias by changing the orientation of a
gene encoding enhanced green fluorescent protein relative
to the origin of replication, and confirmed the previously
observed bias towards the untranscribed strand. We report
that DNA replication can increase the level of
untranscribed strand preference only if that strand also
serves as the lagging strand in DNA synthesis.
Furthermore, the effect of replication on gene repair
frequency and strand bias appears to be independent of
certain mismatched base pairs and oligonucleotide length.
3868
Journal of Cell Science 117 (17)
while maintaining the strandedness of transcription. In E. coli,
this process is RecA independent (Li et al., 2003), differing
mechanistically from the eukaryotic systems, in which the
RecA analogue Rad51 appears to be a crucial component of
the reaction pathway (Liu et al., 2002a; Lundin et al., 2003;
Thorpe et al., 2002; Yanez and Porter, 1999). Nevertheless,
these results prompted us to evaluate the role of DNA
replication in mammalian cells using the well-characterized
SV40 replication system.
Within the cell, the 5.2 kb SV40 genome is contained in a
circular duplex minichromosome with a nucleoprotein
structure analogous to chromatin. Replication initiates at a
well-characterized viral origin through the specific interaction
of the viral protein, large tumour (T) antigen (Deb and
Tegtmeyer, 1987). Replication proceeds bidirectionally, using
both leading- and lagging-strand modes of synthesis. The
SV40 system was tailored for our experiments by creating
four expression vectors (pSK128-eGFP-1, pSK128-eGFP-2,
pSK128-eGFP-3 and pSK128-eGFP-4) derived from pSK128,
a Bluescript plasmid containing an SV40 origin of replication
(Simmons et al., 1998). A mutant enhanced green fluorescent
protein (eGFP) gene was ligated into the pSK128 vector in both
orientations and on different sides of the SV40 origin of
replication so that the effect of either replication polarity and/or
transcriptional status on gene repair could be monitored.
Our plan was to use the SV40 system in order to evaluate
the effect of DNA replication on the strand bias of gene repair.
By varying the orientation of the eGFP gene within the SV40
viral vector, correlations can be made between strand bias as a
function of leading and lagging strand (synthesis) and/or
transcribed and untranscribed strand (expression). We find that
DNA replication accentuates the frequency of gene repair by
directing a strand bias of correction that favours the lagging
strand.
Materials and Methods
Cell line and culture conditions
The COS-1 cell line was obtained from ATCC (American Type
Culture Collection, Manassas, VA) and was grown in Dulbecco’s
modified Eagle’s medium (DMEM) with 4 mM L-glutamine, 4.5 mg
ml–1 glucose, 3.7 mg ml–1 sodium bicarbonate, supplemented with
10% foetal bovine serum at 37°C in 10% CO2.
Plasmid DNA constructs
A single point mutation was created in the eGFP gene of the pEGFPN3 plasmid (Clontech Laboratories) at codon 67 (TAC→TAG) using
a QuikChange Mutagenesis kit (Stratagene). The eGFP cassette
including a cytomegalovirus (CMV) promoter was inserted into
plasmid pSK128 (a gift from D. Simmons, University of Delaware,
Newark, DE) in four different orientations surrounding the origin of
replication. The eGFP gene and CMV promoter were PCR amplified
from the pEGFP-N3 vector (both wild-type and mutant eGFP) using
primers designed to add restriction sites suitable for subcloning the
fragment into the pSK128 vector [Invitrogen high-fidelity polymerase
chain reaction (PCR) supermix]. SpeI and BstXI restriction sites were
added to the eGFP-1 and eGFP-3 constructs and Eco10 and ClaI
restriction sites were added to eGFP-2 and eGFP-4 with the following
primer sets:
eGPF-1F, 5′-GCCTCCACCGCGGTGGGATAACCGTATTACCGCCATGC-3′;
eGFP-1R, 5′-CTTAACTAGTTTGCCGATTTCGGCCTATTGGT-3′;
eGFP-2F, 5′-GCCTATCGATGATAACCGTATTACCGCCATGC-3′;
eGFP-2R, 5′-GCCTGGGCCCCTTGCCGATTTCGGCCTATTGGT3′;
eGFP-3F, 5′-GCCTACTAGTGATAACCGTATTACCGCCATGC-3′;
eGFP-3R, 5′-CTTACCACCGCGGTGGTTGCCGATTTCGGCCTATTGGT-3′;
eGFP-4F, 5′-CTTAGGGCCCCGATAACCGTATTACCGCCATGC3′;
eGFP-4R, 5′-GCCTATCGATTTGCCGATTTCGGCCTATTGGT-3′.
Following PCR amplification, the fragments were isolated, digested
with the appropriate restriction enzymes (New England Biolabs) and
gel purified. The inserts were then ligated into the pSK128 vector.
Plasmids were sequenced to confirm nucleotide alterations.
Oligonucleotides
eGFP3S/72T and eGFP3S/72NT are single-stranded DNA
oligonucleotides
72
nucleotides
long
containing
three
phosphorothioate linkages at each terminus. eGFP3S/72T targets the
transcribed strand of the eGFP gene, and eGFP3S/72NT targets
the untranscribed strand. These oligonucleotides are entirely
complementary to the eGFP gene except for one centrally located
nucleotide designed to convert the TAG stop codon in the eGFP
chromophore region to TAC, encoding tyrosine and resulting in a gene
that expresses functional protein. eGFP-TAT3S/72T and eGFPTAT3S/72NT are identical to eGFP3S/72T and eGFP3S/72NT apart
from the centrally located base, which, in these cases, was designed
to convert the stop codon to TAT (tyrosine). Oligonucleotides
eGFP3S/44T, eGFP3S/44NT, eGFP3S/24T and eGFP3S/24NT are
analogous to eGFP3S/72T and eGFP3S/72NT except that they varied
in length, being 44 (base pairs 14-57 of the 72-mer oligonucleotide)
and 24 (base pairs 24-47 of the 72-mer oligonucleotide) nucleotides
long, respectively. Hyg3S/74T, a noncomplementary oligonucleotide
was used as a nonspecific targeting control (Liu et al., 2001).
Correction of the eGFP gene
Cells were grown to 80% confluence, trypsinized and resuspended in
serum-free medium. Square-wave electroporation (BTX ECM 830; 4
mm gap cuvette, two pulses, 200 V, 20 milliseconds) was used to
introduce 5 µg plasmid and 10 µg oligonucleotide into the COS-1
cells (2×106 cells in a 100 µl volume of serum-free medium). The
electroporated cells were resuspended in complete medium, seeded in
a six-well plate and allowed to recover for 40 hours after
oligonucleotide treatment. The cells were then trypsinized and
resuspended in buffer [PBS, 0.5% bovine serum albumin (BSA), 2
mM EDTA pH 8.0, 2 µg ml–1 propidium iodide (PI)] and assayed
using a FACScaliber flow cytometer (Becton Dickinson, Rutherford,
NJ). Correction was calculated using the CellQuest and GFP/PI
programs. More specifically, the program was set for the appropriate
cell size (forward scatter versus side scatter) and the population of
single cells was gated for analysis. Using the negative control (minus
PI, minus GFP), the background fluorescence was set by positioning
the cells in the tenth decade of the dot plot by adjusting the voltage
for the FL1 (GFP) and FL2 (PI) channels. The machine composition
was then set for multi-fluorochrome experiments using a GFP control
sample containing no PI and increasing the compensation to bring the
signal toward the FL1 parameter. Finally, the last control (PI and no
GFP) was used to increase the compensation to bring the signal toward
the FL2 parameter. Samples of 50,000 cells each were analysed and
cells that were GFP positive and PI negative were scored as corrected
cells. Transformation efficiency was determined by electroporating
wild-type eGFP plasmids identical in gene orientation relative to the
SV40 origin into COS-1 cells and measuring the level of eGFP
expression. The correction efficiency of mutant eGFP plasmids was
then calculated by dividing the percentage of fluorescent cells
resulting from oligonucleotide treatment by the percentage of
Gene repair and gene therapy
fluorescent cells electroporated with the wild-type gene. This control
was part of every experimental design and run in every experiment.
Results
DNA replication affects, but does not change, the
preferential correction of the untranscribed strand
In order to simplify the terminology in this paper, we use the
terms ‘targeted nucleotide exchange’ (TNE) and ‘gene repair’
interchangeably. The basic experimental system contained a
mutant eGFP cassette under the control of a CMV promoter
inserted into a plasmid, pSK128, adjacent to the SV40 origin
of replication (Fig. 1). The mutation in the eGFP gene is a
single base substitution (TAC→TAG), creating a stop codon.
Square-wave electroporation was used to introduce the mutant
eGFP plasmid and oligonucleotide into COS-1 cells. The
single-stranded DNA oligonucleotides are composed of a
single, centrally located noncomplementary base and three
phosphorothioate linkages at each terminus to provide
resistance to nuclease degradation. Correction, as directed
by the oligonucleotide, enables a phenotypic readout of
conversion through expression of the enhanced GFP gene.
Correction events were measured after a 40 hour recovery
period using fluorescence-activated cell sorting (FACS)
analysis.
A series of eGFP reporter constructs was created in order to
examine the influence of replication on the previously
established strand bias of correction, presumably determined
by transcriptional activity. Plasmid pSK128 served as a host
for the new constructs that differ in the orientation of the
pSK128eGFP-1
eGFP-1
5.0Kb
mutant eGFP gene relative to the SV40 origin of replication
(Fig. 2). Essentially, the leading and lagging strands are
reversed relative to the transcribed or untranscribed strand in
this panel of constructs. For example, eGFP-1 and eGFP-2
differ in the combination of strands that function as the leading
or lagging and transcribed or untranscribed; in eGFP-1, the
leading strand is paired with the untranscribed strand etc. This
panel enables us to assess the impact of transcription and
replication on gene repair either independently or jointly. COS1 cells, which express T antigen, are able to support replication
of a SV40-based plasmid and thus were used as the host cells
in these experiments.
The replication assay was initialized by the transformation
of the appropriate plasmid and the oligonucleotide directed to
either the transcribed or the untranscribed strand of the mutant
eGFP gene. The correction reaction was carried out for 40
hours and the converted cells identified by FACS. As shown in
Fig. 3A, the results indicate that each eGFP plasmid harbours
a correction bias favouring the untranscribed DNA strand,
further supporting previous observations that the untranscribed
strand is the preferred target in the gene repair reaction. The
level of transcriptional bias varied between 1.5 and 3 times,
and no replication bias is obvious on initial inspection;
plasmids eGFP-1 and eGFP-4 are more readily corrected on
the leading strand, whereas the lagging strand is corrected
more frequently in plasmids eGFP-2 and eGFP-3. The data
indicating a bias towards the untranscribed strand on each
plasmid target was then re-analysed and recalculated by
dividing the correction obtained with the NT oligonucleotide
by the correction obtained with the T oligonucleotide (Fig. 3B).
Mutant eGFP gene
and CMV promoter
1756 bps
Wild type . . . GTG ACC ACC CTG ACC TAC GGC GTG CAG TGC TTC . . .
. . . CAC TGG TGG GAC TGG ATG CCG CAC GTC ACG AAG . . .
. . . V
T
T
L
T
Y
G
V
Q
C
F . . .
Mutant
3869
. . . GTG ACC ACC CTG ACC TAG GGC GTG CAG TGC TTC . . .
. . . CAC TGG TGG GAC TGG ATC CCG CAC GTC ACG AAG . . .
. . . V
T
T
L
T
*
eGFP3S/72T:
5’ G*T*G* CCC TGG CCC ACC CTC GTG ACC ACC CTG ACC TAC GGC GTG CAGTGC TTC AGC CGC TAC CCC GAC CAC* A*T*G 3’
eGFP3S/72NT:
5’ C*A*T* GTG GTC GGG GTA GCG GCT GAA GCA CTG CAC GCC GTA GGT CAGGGT GGT CAC GAG GGT GGG CCA GGG* C*A*C 3’
Fig. 1. Plasmid target for gene repair. The
mutant eGFP cassette including a CMV
promoter (1756 bp) was inserted into plasmid
pSK128 adjacent to the SV40 origin of
replication. The eGFP gene is shown in blue,
the promoter in green and the SV40 origin of
replication in yellow. The target gene and
SV40 origin of replication are not drawn to
scale. The replacement mutation incorporated
into the eGFP gene occurs at codon 67,
substituting a TAC (tyrosine) for a TAG (stop
codon). This codon is present in the
chromophore region and is essential for
production of the functional protein. The
oligonucleotides designed to direct conversion
of the mutant eGFP gene are 72 nucleotides
long and contain three phosphorothioate
linkages on each end.
3870
Journal of Cell Science 117 (17)
eGFP-1
pSK128eGFP
5.0Kb
eGFP-3
eGFP-2
eGFP-4
2
1
Leading / NT
Lagging / NT
Lagging / T
Leading / T
4
3
Leading / T
Lagging / T
Lagging / NT
Leading / NT
Plasmids eGFP-1 and eGFP-4 are similar in construction, in
that the untranscribed strand correlates with the leading strand
and plasmids eGFP-2 and eGFP-3 are similar in that the
untranscribed strand correlates with the lagging strand of
replication (Fig. 2). When Student’s t test was conducted
comparing the untranscribed strand bias found for plasmids
eGFP-1 and eGFP-4 with the preference found on plasmids
eGFP-2 and eGFP-3, the correction values and their standard
deviations reveal a statistically significant difference, with P
values of 0.002 (Fig. 3B). In short, when the untranscribed
strand is also the lagging strand, as in plasmids eGFP-2 and
eGFP-3, a higher level of correction is observed than when the
untranscribed strand is also the leading strand, as in plasmids
eGFP-1 and eGFP-4. Because the major difference between
these pairs of plasmid targets is that the replication strand is
reversed (the identity as a untranscribed strand is maintained),
we believe that the DNA replication process itself impacts the
strand bias. The highest frequency of repair occurs when the
lagging strand of replication is also the untranscribed strand of
the gene.
Changing the mismatch created by the oligonucleotide
does not alter the favourable NT/lagging strand bias
Both oligonucleotides (eGFP3S/72NT and eGFP3S/72T) are
designed to convert the TAG stop codon to TAC, encoding
tyrosine, but eGFP3S/72NT creates a G-G mismatch with the
untranscribed strand, whereas eGFP3S/72T creates a C-C
mismatch with the transcribed strand. To evaluate the potential
impact of such a discrepancy on the factors that influence the
repair bias outlined above, two new oligonucleotides were
designed that direct the conversion of the mutated TAG codon
to TAT. These oligonucleotides, eGFP-TAT3S/72NT and
Fig. 2. Array of plasmids that serve as target for
gene repair. Three additional targeting vectors
using the base vector outlined in Fig. 1 were
constructed such that each differs in the orientation
of the eGFP gene with respect to the origin of
replication. This reverses the correlation between
the leading and lagging strand of replication and
the transcribed and untranscribed strand within the
eGFP gene. For example, in plasmids pSK128eGFP-1 and pSK128-eGFP-4, the leading strand of
replication is also the untranscribed strand and the
lagging strand is also the transcribed strand,
whereas, in plasmids pSK128-eGFP-2 and
pSK128-eGPF-3, the leading strand of replication
is also the transcribed strand and the lagging strand
is also the untranscribed strand. The eGFP gene is
shown in blue, the CMV promoter in green and the
SV40 origin of replication in yellow. The target
gene and SV40 origin of replication are not drawn
to scale.
eGFP-TAT3S/72T, create a G-A and a C-T mismatch,
respectively, with the designated strand (NT or T). Plasmids,
pSK128-eGFP-1 and pSK128-eGFP-3, were targeted with
these T and NT oligonucleotides to determine whether the
replication influence on transcription bias remains, regardless
of a change in base mismatch. The same strategy was used to
measure the effects of various mismatches on a similar process
in E. coli (Li et al., 2003). As shown in Table 1, the relationship
between transcription and replication strands, favouring the
NT/lagging strand combination in gene repair frequency,
persisted in spite of the alternative mismatch created by eGFPTAT3S/72NT or eGFP-TAT3S/72T.
Oligonucleotide length does not affect the NT/lagging
strand bias but does affect overall frequency
One of the most reproducible effects in gene repair is the
observation that the length of the oligonucleotide influences the
frequency of the reaction (Brachman and Kmiec, 2002; Ellis
et al., 2001; Igoucheva et al., 2001; Liu et al., 2001; Zhang et
al., 2003). In general, the longer the oligonucleotide, the higher
the correction frequency obtained, independent of the
preference for the untranscribed strand. Thus, strand bias and
length dependence were tested in this system using the
standard 72-mer oligonucleotide and two shorter derivatives –
a 44-mer and a 24-mer oligonucleotide. As shown in Fig. 4, a
length dependence was found, confirming other reports, with
the standard 72-mer yielding a higher level of correction events
than the 44-mer; the 44-mer was more effective than the 24mer. Importantly, however, the relationship between T/lagging,
NT/leading, T/leading and NT/lagging strands using plasmids
pSK128-eGFP-1 and pSK128-eGFP-3 remained the same for
all oligonucleotides.
Gene repair and gene therapy
72-mer oligo
A
% Correction
4
leading
7.0
lagging
6.0
3
2
NT
1
T
NT
NT
T
T
T
NT
% correction
5
A
eGFP-1
eGFP-2
eGFP-3
eGFP-4
4.0
NT
3.0
T
2.0
T
0.0
eGFP-1
B
2.5
% correction
Fold change NT vs T
NT
lagging
5.0
Target Plasmids
3.0
leading
1.0
0
3.5
3871
2.0
1.5
1.0
0.5
0.0
eGFP-1
eGFP-2
eGFP-3
eGFP-4
7.0
B
6.0
5.0
4.0
3.0
2.0
1.0
0.0
eGFP-3
44-mer oligo
NT
NT
T
T
eGFP-1
eGFP-3
Target Plasmids
24-mer oligo
Table 1. Alternative mismatch does not affect the strand
bias of gene repair
Strand
Correction (%)*
Plasmid
Transcription
Replication
TAC†
TAT‡
pSK128-eGFP-1
pSK128-eGFP-1
pSK128-eGFP-1
pSK128-eGFP-3
pSK128-eGFP-3
Nonspecific§
–
Lagging
Leading
Leading
Lagging
0.01±0.02
1.48±0.25
2.26±0.69
1.27±0.32
3.52±0.76
0.01±0.03
1.85±0.40
2.39±0.41
0.84±0.22
5.60±1.91
T
NT
T
NT
*The correction efficiencies obtained with each type of oligonucleotide
were calculated as stated in Materials and Methods.
†Oligonucleotides designed to convert the mutant TAG codon to TAC,
encoding tyrosine, directed to the T and NT strands.
‡Oligonucleotides designed to convert the mutant TAG codon to TAT,
encoding tyrosine, directed to the T and NT strands.
§The vector pSK128-eGFP-1 was also targeted with a nonspecific
oligonucleotide, Hyg3S/74NT, to evaluate background (nonspecific) levels of
eGFP correction.
Abbreviations: NT, untranscribed strand; T, transcribed strand.
7.0
6.0
% correction
Fig. 3. Strand bias in gene repair is influenced by both transcription
and replication. (A) Each of the four mutant eGFP targeting vectors
pSK128-eGFP-1 to pSK128-eGFP-4 was electroporated into COS1 cells together with an oligonucleotide designed to target either
the transcribed or the untranscribed strand. The strand of
replication to which each relates is also listed. Targeting with a
nonspecific oligonucleotide resulted in a background of 0.03% or
less by FACS analysis (data not shown). This experiment was
repeated four times and the standard deviations are indicated.
(B) The data accumulated in section A were reanalysed: the
correction frequency obtained using the NT oligonucleotide was
divided by the correction frequency obtained using the T
oligonucleotide for each of the four plasmids. The NT strand
correlates to the leading strand in plasmids 1 and 4, and to the
lagging strand in plasmids 2 and 3. The relative bias towards the
NT strand in plasmids 2 and 3 was found to be significantly
different from the bias towards the NT strand in plasmids 1 and 4
by using Student’s t-test with a P value of 0.002.
C
5.0
4.0
3.0
2.0
1.0
NT
T
NT
T
0.0
eGFP-1
eGFP-3
Fig. 4. The NT/lagging strand bias is unaffected by the length of the
oligonucleotide in the gene repair reaction. pSK128-eGFP-1 and
pSK128-eGFP-3 targeting plasmids were electroporated into COS-1
cells with either the T or NT oligonucleotides (72 bp, 44 bp and 24
bp). Targeting data for the strands of transcription and replication are
indicated for each oligonucleotide length. This experiment was
repeated four times and standard deviations are indicated.
Discussion
The gene repair reaction is initiated with the binding of an
oligonucleotide specific to the target sequence and the
recruitment of cellular proteins, which then catalyse nucleotide
exchange. We believe that the rate-limiting step of this reaction
is the hybridization of the oligonucleotide to the target DNA,
a step largely regulated by the availability of the DNA target.
For example, an actively transcribed region is more accessible
to oligonucleotide binding because it provides an environment
in which the chromatin structure surrounding the gene is
loosened (Igoucheva et al., 2003; Liu et al., 2002b). A
decondensed chromatin structure is known to be more prone
to homologous DNA pairing events necessary for both
recombination and targeted nucleotide exchange (Kotani and
Kmiec, 1994). The importance of transcription in gene repair
was first defined in S. cerevisiae using an inducible promoter
on the target gene. Under transcriptional repression, a low level
of correction was seen, whereas a higher level of gene
3872
Journal of Cell Science 117 (17)
correction was measured when the template was active (Liu et
al., 2002b). Experiments in mammalian cells with a LacZ gene
under the control of the Tet-on and Tet-off promoter confirmed
this observation as an actively transcribed gene was again
found to be repaired at a higher frequency than inactive targets
(Igoucheva et al., 2003). DNA replication can also lead to an
altered or perhaps even disrupted chromatin structure, which
could create an environment that is more amenable for
oligonucleotide pairing and gene repair (Morales et al., 2001).
The influence of transcription on strand bias has been well
documented; oligonucleotides designed to hybridize to the
untranscribed strand of the target result in a higher level of
correction than when the transcribed strand is similarly
targeted (Y. Hu, L. Liu, L. Ferrara and E.B.K., unpublished)
(Igoucheva et al., 2001; Igoucheva et al., 2003; Liu et al., 2001;
Liu et al., 2002b; Nickerson and Colledge, 2003). The basis
for this transcriptional bias has not been fully elucidated but
one explanation was suggested by in vitro modelling
experiments demonstrating that an active T7 phage RNA
polymerase can displace the oligonucleotide from the
transcribed strand of DNA but not the untranscribed strand (Liu
et al., 2002b).
Strand bias favouring the untranscribed strand is not
universal, however, because studies reported by Sherman and
colleagues (Yamamoto et al., 1992b) with the CYC1 gene of S.
cerevisiae revealed a preference for the template or transcribed
strand of DNA. Recently, we re-examined gene repair on this
gene (Brachman and Kmiec, 2003), targeting five different
cyc1– strain variants containing mutations at a codon
downstream of the initial mutation site. Four out of the five
cyc1– strains revealed a preferential targeting mode for the
untranscribed strand but one strain, YMH53, paralleled the
early studies, revealing a transcribed strand bias. Further
examples of a template preference have appeared in episomal
targeting of the LacZ gene in CHO cells in which two
mutations approximately 900 bp apart exhibited opposite
strand biases (Sorensen et al., 2003). In addition, chromosomal
targeting of the mouse dystrophin gene yielded the more
commonly observed untranscribed strand bias in vitro, but in
vivo studies revealed a transcribed strand bias (Bertoni et al.,
2003; Bertoni and Rando, 2003). Studies such as these and
others (Lu et al., 2003) make it clear that, although DNA
transcription might be a major determinant of strand bias, it is
not the only process controlling this choice in the reaction
pathway.
Up to this point, the influence of DNA replication on
targeted nucleotide exchange using modified oligonucleotides
had not yet been examined in detail. In a related study,
however, DNA replication in prokaryotes was found to have a
significant effect when a 70-mer unmodified oligonucleotide
was used to direct homologous recombination in E. coli
(Ellis et al., 2001). Examination of oligonucleotide-mediated
recombination at five genes on the chromosome indicated a
preference correlating not to transcription but to the lagging
strand of DNA replication. More recent data from the same
group (Li et al., 2003) indicate an absolute relationship
between DNA replication and strand bias, and these workers
conclude that transcription plays no part in determining strand
bias in an E. coli system. Such a restriction might be unique to
E. coli, because yeast, and now mammalian cell, strand bias in
gene repair appears to be governed by multiple processes
(Bertoni and Rando, 2003; Brachman and Kmiec, 2003;
Igoucheva et al., 2001; Igoucheva et al., 2003; Liu et al., 2001;
Liu et al., 2002b; Lu et al., 2003; Nickerson and Colledge,
2003; Sorensen et al., 2003). This might reflect an increased
complexity in eukaryotes compared with prokaryotes or the
fact that the E. coli reaction takes place in the absence of the
DNA recombinase RecA. By contrast, eukaryotes display a
dependence on the RecA homologue Rad51 (Liu et al., 2002a;
Lundin et al., 2003; Thorpe et al., 2002; Yanez and Porter,
1999).
Here, we tested a bank of target plasmids containing various
combinations of transcribed and untranscribed strands of the
mutant eGFP gene in a construct in which they were either the
leading or the lagging strand of replication. This system has
allowed us to contrast the impact of replication and the
influence of transcription. Independent of the replication
polarity of each strand, the target plasmids were preferentially
corrected on the untranscribed strand (Fig. 3A). However, the
degree of strand bias appears to be influenced by both
transcription and replication, processes that can occur
simultaneously within the same region of the chromosome. A
statistically significant increase in the untranscribed strand bias
occurs when it also serves as the lagging strand of the
replication process (Fig. 3B). The impact of a replication bias
presents itself most obviously as an enhancer of the
transcriptional bias inherent in the correction process of the
mutant eGFP gene. One effect of the lagging strand serving as
the template strand for gene repair is that it can also reduce the
numerical differences between the strand bias ascribed to the
transcribed and untranscribed strands. The transcriptional bias
seen in plasmids eGFP-1 and eGFP-4 is relatively small
compared with that seen on other target genes and cell lines.
The reason for this reduced transcriptional bias is probably a
competitive effect between transcription and replication,
because the link between the untranscribed and lagging strands
is uncoupled and each might effectively ‘compete’ for the
oligonucleotide. It should be realized that our experiments are
carried on a template with a reporter gene that is heavily
transcribed from a strong promoter. ‘Natural’ genes might not
be transcribed at such a high rate and thus replication might
exert an even greater influence on strand bias under these
conditions.
The influence of mismatch specificity in gene repair has also
been examined most recently in a yeast system (Brachman and
Kmiec, 2003) but the formulation of a complete mismatch
hierarchy is problematic because assay readout is dependent on
transcription of the gene, limiting mismatch choices (Li et al.,
2003). So, mismatches cannot be tested at random or in such
a way that genetic readout or gene function is disrupted. In our
system, the stop codon within the chromophore region of the
mutant eGFP gene must be converted to a tyrosine in order to
maintain the production of a functional protein. The NT oligo
which directs conversion to a TAT produced a slightly higher
level of correction than the NT oligo directing conversion to a
TAC codon. This might indicate that a G/A mismatch is
favoured over a G/G mismatch, but these do not exhibit a
statistical difference. Although only two mismatch types were
examined on each DNA strand, it appears in this restricted
system that mismatch discrimination plays no significant role
in determining strand bias compared with the forces exerted by
either DNA transcription or replication or both (Table 1).
Gene repair and gene therapy
Finally, oligonucleotide length was examined by comparing
correction frequencies obtained using a 72-mer, a 44-mer, or a
24-mer oligonucleotide directed to either the transcribed or
untranscribed strand. An oligonucleotide length response was
observed on the overall frequency of gene repair in that the 72mer directed more correction events than the 44-mer, whereas
the 24-mer led to the fewest conversion events. Consistent with
our previous observation (Liu et al., 2002b), each of these
oligonucleotides exhibited a transcription bias favouring the
untranscribed strand. Furthermore, the enhanced bias effect
of the lagging strand of replication when identical to the
untranscribed strand was also observed for each
oligonucleotide length variant (Fig. 4).
DNA replication can now be added to a list of cellular
processes that influence the process of gene repair.
Transcription was found previously to have a decisive effect on
strand bias and could even mask the influence of DNA
replication on strand preference. However, we do find a role
for DNA replication in creating a strand bias of gene repair,
increasing the frequency of repair when the lagging strand of
the replication fork is the untranscribed strand of the gene.
Other influences on the strand bias of correction will probably
appear as mechanistic studies continue.
We thank D. Simmons for providing the pSK128 plasmid used in
this work and also for helpful discussions. We also thank fellow
laboratory members for useful critiques of the data and the
conclusions drawn. And we are most grateful to S. Engstrom for
manuscript preparation and editorial assistance. Support from NIH
(R01 CA89325) is acknowledged.
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