Transformation with Oligonucleotides Creating
Clustered Changes in the Yeast Genome
Gina P. Rodriguez1, Joseph B. Song1, Gray F. Crouse1,2*
1Department of Biology, Emory University, Atlanta, Georgia, United States of America, 2Winship Cancer Institute, Emory University, Atlanta, Georgia, United States of
We have studied single-strand oligonucleotide (oligo) transformation of yeast by using 40-nt long oligos that create
multiple base changes to the yeast genome spread throughout the length of the oligos, making it possible to measure the
portions of an oligo that are incorporated during transformation. Although the transformation process is greatly inhibited
by DNA mismatch repair (MMR), the pattern of incorporation is essentially the same in the presence or absence of MMR,
whether the oligo anneals to the leading or lagging strand of DNA replication, or whether phosphorothioate linkages are
used at either end. A central core of approximately 15 nt is incorporated with a frequency of .90%; the ends are
incorporated with a lower frequency, and loss of the two ends appears to be by different mechanisms. Bases that are 5–
10 nt from the 59 end are generally lost with a frequency of .95%, likely through a process involving flap excision. On the 39
end, bases 5–10 nt from the 39 end are lost about 1/3 of the time. These results indicate that oligos can be used to create
multiple simultaneous changes to the yeast genome, even in the presence of MMR.
Citation: Rodriguez GP, Song JB, Crouse GF (2012) Transformation with Oligonucleotides Creating Clustered Changes in the Yeast Genome. PLoS ONE 7(8):
Editor: David T. Kirkpatrick, University of Minnesota, United States of America
Received May 31, 2012; Accepted July 12, 2012; Published August 14, 2012
Copyright: ? 2012 Rodriguez, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health [R01 GM80754 to GFC]. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
It was first demonstrated in the Sherman lab that single-
stranded oligonucleotides (oligos) could be introduced into yeast
cells and create permanent changes in the genome, although the
mechanism of that transformation was not understood [1–3].
Later work in both E. coli and yeast, in which events can be
determined in relation to known origins of replication, has shown
that transformation is more efficient when oligos anneal to the
lagging strand [4,5]. Our work [5,6] as well as others  strongly
supports the view that oligo transformation is generally due to the
incorporation of the oligo at the replication fork and the oligo then
serves as a primer for continued replication.
The early experiments used strains proficient in mismatch
repair (MMR). One issue has been the role of MMR in the
transformation process. MMR recognizes DNA mismatches
created in the process of replication and removes bases on the
primer strand, using some sort of strand discrimination process
such as nicks on the newly replicated strand to determine which
strand to remove [8–10]. There are two recognition complexes in
most eukaryotes, MutSa, a heterodimer of Msh2 and Msh6 that
recognizes base-base mismatches and small loops, and MutSb, a
heterodimer of Msh2 and Msh3 that recognizes loops [8–10].
MMR is also important in preventing recombination between
non-identical DNA [8–10]. Although that process of heteroduplex
rejection is not coupled to replication , the invading strand is
preferentially eliminated. Because the function of MMR is to
remove newly replicated DNA or invading DNA that would create
mismatches with the existing DNA, one might have expected
MMR to interfere with the process of oligo transformation.
However, it was postulated that the transformation process in
yeast was a gene correction event requiring MMR  and later
work indicated that Msh2 assisted transformation in yeast, but
hindered transformation in mammalian cells . In contrast,
experiments in E. coli [4,14] and our experiments in yeast  both
demonstrate a strong blocking effect of MMR, consistent with its
activity both in preventing replication errors and heteroduplex
rejection. There seems to be general agreement that oligo
transformation in mammalian cells is blocked by MMR [7,15,16].
With a few exceptions [17–19], experiments with oligo
transformation in eukaryotes have examined only the change of
contiguous bases, mediated usually by bases in the center of a
transforming oligo. Early work from the Sherman lab found that
transformation decreased as the number of central mismatches
increased, except for a situation in which there were 9 mismatches
in a row . That puzzling result can now be understood as the
relative invisibility of a large loop to MMR [5,16]. The Sherman
lab also found that mismatches at either end of an oligo were not
incorporated . One study in yeast found that sites 4 bases apart
were usually incorporated together, whereas a separation of 15 or
more bases apart led to low frequencies of simultaneous
incorporation . A report of oligo transformation in mamma-
lian cells found that sites separated by 14 bases could be
simultaneously incorporated but that the percentage of co-
incorporation appeared to decrease linearly with distance .
Our interest in this study was to better understand the
mechanism of incorporation of oligos into the genome and in
particular the pattern of incorporation throughout the length of
the oligo. There could well be biases in 59 versus 39 end
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incorporation, and on whether MMR were present and on which
strand of replication an oligo were incorporated. One of our future
interests is to use oligos to incorporate specific damaged bases into
the genome for subsequent analysis, and for such studies it would
be particularly important to know what regions of an oligo were
likely to be incorporated. In addition, there has been considerable
interest in oligo transformation as a means of gene therapy, and
understanding the process is crucial to its use .
Materials and Methods
The trp5 G148Cm mutation was created as described for the
other trp5 mutations  using delitto perfetto  and created the
sequence CGATGTTATCCAACTGGGA starting at position
138 of TRP5 with mutated bases underlined. The location of the
TRP5 gene in both orientations with respect to the surrounding
genes and the nearby ARS306 origin of replication is illustrated in
Fig. 1. The lys2CT1265GA mutation was similarly created by delitto
perfetto. The genotypes of strains used in these experiments is given
in Table S1. All gene deletions were created by one-step disruption
with PCR generated fragments. In general gene deletions were
made from a PCR fragment generated from the collection of yeast
gene deletions . The kanMX4 resistance marker was changed
to hphMX4 by transformation with a fragment from pAG32 .
Transformation was as described previously . For a typical
transformation, 200 pmol of a Trp oligo and 200 pmol of
LYS2TCARev40 was used for a 200 ml of this cell suspension.
Immediately after electroporation, the cell suspension was added
to 5 ml YPAD, and the cells incubated at 30u with shaking for 2 h.
Cells were then centrifuged, washed with H2O, and plated on
synthetic dextrose (SD) medium lacking either tryptophan or lysine
 to select transformants. The Lys oligo was originally designed
to be used as an internal control for transformation. However, it
was subsequently found that the number of Lys+ transformants
was not correlated well enough with the number of Trp+
transformants to be used as an internal control (results not shown).
The sequence of all oligos used is given in Table S2.
PCR and Revertant Analysis
Individual Trp+ revertants were picked into 200 ml SD-Trp
medium in 96-well deep-well plates, grown overnight at 30u with
shaking, a small aliquot of each transferred to fresh SD-Trp
medium with a Boekel Microplate Replicator and grown overnight
again, and finally transferred with the replicator to another deep-
well plate for overnight growth in 300 ml YPAD. Cells were then
transferred with the replicator to a PCR microplate containing
15 ml per well of 2 mg/mL Zymolyase 20T (USBiological) in
0.1 M Phosphate Buffer pH 7.4 and incubated at 37u for 30 min
and 95u for 10 min. After incubation, 85 ml H2O was added to
each well. PCR was performed using 5 ml of the lysate in a total
volume of 50 ml of the recommended buffer with 0.3 mM trpseq2
and trpseq8 primers and 0.5 ml Takara e2TAK DNA polymerase
for 30 cycles at 56u C. For restriction digestion, 5 ml of the PCR
reaction was incubated with 2 units of SphI (New England Biolabs)
in the recommended buffer in a total volume of 15 ml at 37u
overnight and analyzed by gel electrophoresis. Sequencing of PCR
products was performed by Beckman Coulter Genomics.
Measuring incorporation of multiply-marked oligos
Even under optimal conditions in the absence of MMR, the
frequency of oligo transformation is so low that one needs to be
able to select for those cells that have successfully incorporated the
oligo. We turned to the set of trp5 point mutations we previously
constructed . These strains contain a mutation at either
nucleotide position 148 or 149 of the TRP5 gene and can only be
reverted to the wild type phenotype by restoring the original TRP5
sequence and thus have an extremely low rate of spontaneous
reversion . A potential problem was that the region
surrounding the mutant base is highly conserved, constraining
the location of any changed base. We therefore introduced several
mutations into the region, maintaining with one exception the
original amino acid sequence, but increasing the number of
positions into which a different base could be substituted (Fig. 2).
Because this mutant trp5 G148Cm gene is placed close to a
dependable origin of replication, and is present in both
orientations relative to the origin (Fig. 1), we know which strand
is replicated as leading and which as lagging and can reverse the
replication strands by using a strain of opposite TRP5 orientation
For transformation, we used an oligo (N) that would create 7
mismatches when annealed to the G148Cm region (Fig. 2). In
addition, in many cases oligos for transformation have been
synthesized with phosphorothioate linkages at the ends for the
purpose of increasing oligo stability in the cell , and so we also
tested the effect of such linkages on marker incorporation, using
oligos with 4 phosphorothioate linkages on the 59 (59 4P) or 39 (39
4P) ends to determine if phosphorothioate linkages would increase
incorporation of nucleotides on the ends of the oligos. Because the
oligos created only base-base mismatches in the genome, only
MutSa should recognize the incorporated oligos. Therefore
experiments were accomplished by transforming strains proficient
(wt) or deficient (msh6) in MutSa, with individual revertants then
Mismatch repair has little effect on the pattern of oligo
Transformation of Oligo N was much more efficient into msh6
strains deficient in MMR compared to MMR-proficient strains,
but our interest was in determining what portions of the oligo were
incorporated, rather than the absolute efficiency. The results of
transforming Oligo N into msh6 and wild-type G148Cm strains in
both F and R orientations are shown in Fig. 3. All revertants
included the C at position 21 of the oligos, as that is essential for a
Trp+ phenotype. It was also evident that under any condition,
bases within about 10 nt of that central position were incorporated
at a high frequency, but generally less than 100%, and that the
ends of the oligos were much less frequently incorporated.
Sequencing also revealed that, as expected, there was no
‘‘skipping’’ of incorporated nucleotides (data not shown). The
Figure 1. Location of TRP5 gene in G148Cm F and R strains. The
TRP5 gene, in both orientations, replaced the RNQ1 gene on
Chromosome III near the ARS306 origin of replication as illustrated
above. Shown below is a scale of distance in kb.
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number of Trp+ revertants was higher with oligos annealing to the
lagging strand (R) (data not shown), but as can be seen in Fig. 3 the
pattern of incorporation was in general the same in both
orientations. In addition, the pattern of incorporation wassimilar
in the presence or absence of MMR (Fig. 3).
The effect of phosphorothioate linkages on
The loss of sequences from both ends of the oligo raised the
question of whether the loss was due to exonucleolytic degradation
that could be prevented by the use of phosphorothioate linkages
that have been used in other oligo transformation experiments
. We therefore tested the effect of phosphorothioate linkages
on marker incorporation, using oligos with 4 phosphorothioate
linkages on either end. The results are shown in Fig. 4A and B.
Somewhat surprisingly, the presence of phosphorothioate linkages
at the 59 end of the oligo made no noticeable difference in
retention of sequences at the 59 end of the oligo. However, the
retention of nucleotides at the 39 end of the oligo was modestly
increased (Fig. 4A; compare to Fig. 3). The presence of
phosphorothioate linkages at the 39 end of the oligo also made
no difference in retention of sequences at the 59 end. However, at
the 39 end in wild-type strains, there was a noticeable increase in
retention of nucleotides, but not in msh6 strains (Fig. 4B). For
example, retention of the marker 33 nt from the 59 end was 87%
for the modified and 68% for the unmodified in F wild-type
strains, and 96% compared to 72% in R strains.
Incorporation of 59 oligo nucleotides is influenced by
If oligos serve as primers for replication, then an upstream
Okazaki fragment on the lagging strand, or the replicating end on
the leading strand, would have to eventually join up with the oligo-
primed fragment and be ligated together. This reaction usually
involves formation of a flap on the 59 end of the primed fragment
, and one explanation for the loss of nucleotides on the 59 end
of the oligos, and the lack of protection of the 59 end by
phosphorothioate linkages, was that the ends were being lost due
to flap cleavage. The flap is usually cleaved by the Fen1
endonuclease (encoded by the RAD27 gene in yeast) ;
transformation by oligo N was therefore examined in rad27 msh6
strains. As observed in Fig. 3, elimination of Rad27 led to
increased retention of the 59 ends of oligos on both the leading and
lagging strands of replication (22% vs. 7% and 32% vs. 6% at nt 6
from the 59 end for F and R strains respectively), indicating a role
for Rad27 in loss of the ends.
Mismatch repair processing of mismatches
The fact that the pattern of incorporation of oligo N was the
same in the presence or absence of MMR suggested that those few
oligos that escaped the action of MMR escaped completely. We
Figure 2. Sequences of TRP5 mutant regions and oligonucleotides used for reversion analysis. The first line shows the wild-type
sequence of TRP5 from position 128 and the second line shows the G148C sequence, with the mutant C highlighted in yellow that must revert to G
for TRP5 function . The G148Cm mutant contains several changes (highlighted in green) designed to create additional completely degenerate
third codon positions, with those of interest underlined. Oligo N creates 7 mismatches (highlighted in yellow and blue) upon annealing with the
G148Cm sequence, whereas Oligo G creates the indicated 3, and Oligo TG, 4 mismatches. The G in the oligos 12 nt from the 59 end when
incorporated creates a new SphI restriction site.
Figure 3. Nucleotides flanking the base inducing Trp+ + rever-
sion are usually incorporated in Trp+ + revertants. Oligo N of Fig. 2
was transformed into G148Cm strains of the indicated genotype and
Trp+ revertants sequenced to determine which oligo-induced muta-
tions were present in each revertant, with the distance indicated from
the 59 end of the oligo. The indicated percentages are determined from
approximately 48 revertants of each genotype. Trp+ revertants
containing only the mutation at position 21 were assumed to be
spontaneous revertants and were observed, rarely, and were not
counted. In all experiments, oligos anneal to the leading strand of
replication in the F orientation, and to the lagging strand in the R
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designed a different oligo, oligo G, with only a few mismatches
located centrally, illustrated in Fig. 2. That oligo creates a C-C
mismatch to induce Trp reversion, a G-A mismatch 8 nt 59 that
would create a new SphI restriction site with retention of that part
of the oligo, and a G-G mismatch between those two positions.
The equivalent portion of oligo N is retained 91-94% of the time
in both MMR-proficient and deficient strains, and we expected
the same for oligo G. For oligo G, instead of sequencing, we could
measure retention by SphI restriction site analysis of Trp+
revertants. As shown in Fig. 5A, strains deficient in MutSa all
incorporated close to 100% of the central portion of oligo G.
However, in wild-type strains or strains lacking only MutSb (msh3
strains), incorporation of that region varied from 64–79%. As
illustrated in Fig. 6A, a revertant that does not have the SphI site
but is Trp+ must have lost sequence from the 59 end of the oligo
during the initial round of replication. A likely explanation is that
MMR recognizes the G/A and G/G mismatches, but recognizes
the C/C mismatch more poorly , and in 25% of the cases,
MMR-directed excision does not proceed through the C/C
mismatch. In order to test this hypothesis, wild-type strains were
transformed with Oligo TG (Fig. 2) that would create an
additional mismatch 39 of the C-C mismatch. With the additional
mismatch created by Oligo TG, all Trp+ revertants contained the
SphI site (Fig. 5B). Thus either the entire central portion of the
oligo escapes MMR, or as illustrated in Fig. 6B, all of the
mismatches are removed.
The use of oligos creating multiple mismatches with the genome
has revealed valuable information, not only for their subsequent
use, but also about replication and mismatch repair. If oligos serve
as primers for replication, one might have expected them to
transform only when annealed to the lagging strand of replication,
which is replicated discontinuously, and not to the leading strand
which is presumably replicated in a continuous fashion. However
Figure 4. Phosphorothioate linkages make little difference in
retention of oligo end sequences. Oligo N was transformed and
results analyzed as in Fig. 3. (A) phosphorothioate linkages between the
4 nucleotides at the 59 end of Oligo N. (B) phosphorothioate linkages
between the 4 nucleotides at the 39 end of Oligo N.
Figure 5. A C-C mispair is not always recognized by MMR. (A)
Strains of the indicated genotypes in either the F or R orientation were
transformed with Oligo G, and Trp+ revertants were selected and
scored for the percentage that contained an SphI site created by a
nucleotide in the oligo 8 nt 59 of the C creating the Trp+ phenotype
(Fig. 2). At least 40 revertants were scored in each phenotype. The error
bars indicates the standard deviation observed from 2 independent
experiments. (B) Wild-type strains were transformed with Oligo TG and
analyzed as in (A). Data for Oligo G are from (A). Oligos anneal to the
leading strand of replication in the F orientation, and to the lagging
strand in the R orientation.
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as we have found in yeast  and others in E. coli , although
transformation is more efficient with oligos targeted to the lagging
strand, it occurs with oligos targeted to the leading strand a few
fold less in yeast and 30-fold less in E. coli. Although the efficiency
of transformation is greatly reduced by MMR , the pattern of
oligo incorporation is generally very similar in the presence or
absence of MMR and whether the oligo was targeted to the
leading or lagging strand of replication (Figs. 3 and 4). In all
conditions examined, there was a clear asymmetry of retention of
ends of the oligo, with the 59 end being much less likely to survive
than the 39 end, as can be seen in Figs. 3 and 4. These results
suggest that the mechanism of incorporation is independent of
MMR, is similar on the leading and lagging strands of replication,
and that usually all mismatches are recognized and eliminated by
MMR, or none are.
One issue that arises from these results is the degree to which
the leading strand is replicated in a continuous fashion. The DNA
polymerase that replicates the leading strand, Pol e, has at least in
vitro a processivity that is not any greater than Pol d, the
polymerase that replicates the lagging strand . In addition,
there is considerable evidence both in E. coli  and in yeast 
that replication on the leading strand can also be discontinuous.
Thus it is perhaps not surprising that oligo transformation can
occur on the leading strand. A large amount of evidence supports
the view that lagging strand synthesis is done by Pol d and that
leading strand replication is initially carried out by Pol e . A
recent model proposes that synthesis after any interruption on the
leading strand is completed by Pol d . In that context it would
be extremely interesting to know which polymerase was respon-
sible for elongation of oligos targeted to the leading strand.
The central core of the oligo was usually incorporated, but the
59 end was rarely incorporated and about 1/3 of the time, 10 or
more nucleotides on the 39 end were not incorporated. The loss of
nucleotides from the two ends appears to occur by fundamentally
different mechanisms. Phosphorothioate linkages on the 59 end of
the oligo make no difference in the pattern of loss of the 59 end,
suggesting that the nucleotides are not removed exonucleolytically,
or that the enzymes involved are not affected by the altered
linkages. There is considerable evidence that phosphorothioate
linkages do protect against a number of exonucleases , and so
the lack of effect suggests that the 59 end loss is not exonucleolytic.
If the oligos serve as primers for replication, then ultimately the
DNA primed by the oligo would have to be joined to DNA
synthesized upstream as in normal Okazaki fragment maturation
[25,31]. Thus the loss of the 59 end sequences could be due to the
formation of a flap at the 59 end with subsequent excision of the
flap by Rad27 [25,31,32]; the increase in 59 end sequences in a
rad27 strain indicates a role for Rad27 in flap excision of oligo
sequences (Fig. 3). There are alternate pathways for fragment
maturation not involving Rad27 ; the combination of those
pathways is likely responsible for the major loss of oligo 59
sequences, on both the leading and lagging strands. Although the
leading strand of replication is generally replicated in a continuous
manner, these results also indicate that new priming events on the
leading strand are processed similarly to those on the lagging
strand. It may be indicative of some difference between the two
strands that on the leading strand the absence of Rad27 appears to
Figure 6. Models for action of MMR on oligos G and TG. (A) Oligo G anneals and primes replication as indicated. MMR should be the only
system that would recognize the mismatches, and any nucleotides that remain after completion of the first round of replication should be replicated
in the second round. If the C-C mismatch were poorly recognized by MMR, it is possible that it could remain after MMR action, as illustrated. (B) Oligo
TG anneals and primers replication as indicated. In this case, even if the C-C mismatch were poorly recognized, MMR action should ensure removal of
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effect the incorporation of some internal nucleotides in a manner
different from that observed on the lagging strand (Fig. 3, compare
incorporation of nucleotides 12 and 15 from the 59 end in rad27
msh6 F and R strains). A surprise is that although phosphorothioate
linkages on the 59 end do not make any appreciable difference in
retention of the 59 end, they do appear to have some affect on
retention of the 39 end (for the nucleotide at position 33, 89%
retention versus 79% in the R orientation, and 80% vs. 71% in the
F orientation) (Fig. 7). If the 59 end were lost solely due to an
endonucleolytic flap cleavage 5–10 bases from the 59 end, one
would not expect phosphorothioate linkages in the 59-most 4
nucleotides to have any effect on maintenance of 39 end sequences.
Phosphorothioate linkages do offer some protection on the 39
end in wild-type, but little if any in msh6 strains (Fig. 4B compared
to Fig. 3). What could account for the loss of 39 end nucleotides in
MMR-proficient strains that is not observed when there are
phosphorothioate linkages at the 39 end? It has been demonstrated
that phosphorothioate linkages protect against at least some DNA
polymerase proofreading activities . This suggests that some 39
nucleotides may be lost by MMR-directed excision from the 39
end, possibly by the 39 proofreading exonuclease of the replicating
polymerase . This could be an indication of a limited type of
MMR function, involved in only surveillance of the 39 end, as
there is no overall difference in pattern of unmodified oligos with
or without MMR (Fig. 3). The 39 end of transforming oligos is not
always lost, as we have been able to induce transformants using
oligos in which the 39 nucleotide has to be incorporated for
reversion; this process can be quite efficient when the terminal
mismatch is well tolerated, such as an 8-oxoG-A mismatch (results
There have been different conclusions on the use of phosphor-
othioate bonds in oligos used for transformation. The original
experiments on transformation in yeast used unmodified oligos [1–
3]. Phosphorothioate linkages were later found to increase
transformation in yeast by several fold in a different lab . In
mammalian cells, the situation is complex . The use of oligos
protected with phosphorothioate linkages at both ends induced cell
cycle arrest and double-strand breaks [34,35]. In one study it was
found that oligos with phosphorothioate linkages at both ends gave
greater transient correction than oligos with unmodified ends, but
gave significantly fewer stable colonies . In the absence of
MSH2, transient correction was highest for unmodified oligos,
followed in decreasing order by oligos modified at the 39, 59, or
both 39 and 59 ends, but for those experiments relative viable
formation of colonies was not reported . More recently it was
found that transformation of msh2 cells was more efficient with
unmodified oligos than oligos with phosphorothioate linkages at
both ends and that the unmodified oligos created much less cell
cycle disturbance . That would suggest that, at least in
mammalian cells, oligos with phosphorothioate linkages at the
ends can lead to double-strand breaks and cell cycle disruption and
therefore fewer viable transformed colonies than the use of
unmodified oligos, although the reason for the difference was not
understood . A recent report studying oligo transformation in
HeLa cells found that toxicity was correlated with increasing
number of phosphorothioate bonds, possibly due to stimulation of
cellular immunity, and that a few internal phosphorothioate
linkages 39 to the mismatch were most effective in creating stable
What might be the cause of cell-cycle arrest and double strand
breaks observed in mammalian cells due to oligo transformation
with oligos containing phosphorothioate linkages? Based on our
observations, it appears that phosphorothioate linkages on the 59
end could be problematical in replication fragment joining. It is
clear that whatever process is used to join the 59 end of the oligo
into the completed replicated strand involves some sort of 59 end
processing, and the fact that a change in such processing could
cause even slight differences on incorporation of 39 end sequences,
as observed in Fig. 7, suggests a significant change in oligo
incorporation. Our experiments do not measure the incorporation
of bases at the very 39 end of the oligo, but it is clear that there is a
tendency to lose bases at the 39 end, and it may be that the usual
method of primer extensive could involve a small degree of 39
resection, which would be prevented by phosphorothioate
linkages, again partially disrupting the normal incorporation.
The interesting exception to a similar pattern of transformation
in wild-type and msh6 strains was provided by oligo G, where in
wild-type, but not msh6 strains, nucleotides close to the center of
the oligo were lost in 25% of transformants (Fig. 5A). This loss was
shown to be likely due to the occasional failure of MMR to
recognize a C-C mismatch, as a well-recognized mismatch created
just to the 59 side of the C-C mismatch (oligo TG) resulted in
retention of all oligo nucleotides in Trp+ revertants (Fig. 5B). The
location of the excised nucleotides relative to the retained
nucleotides showed that in this case MMR-directed excision was
from the 59 end of the oligo and that excision must not have
proceeded more than 4 nucleotides past the recognized mismatch-
es or else as can be seen in Fig. 6A, the C-C mismatch would have
been removed resulting in no Trp+ revertants. Recent work
analyzing single-base mispairs created by polymerase errors found
that errors created by Pol a were corrected more efficiently by
MMR than errors created by Pol d, and it was hypothesized that
the difference might be due to the use of the 59 end of the Okazaki
fragment as a strand discrimination signal . Our results are
consistent with MMR-directed excision from the 59 end of a
replicating segment, and further indicate that such excision likely
stops directly after the recognized mispair.
In order to make optimum use of oligo transformation, it is
important to understand the parameters of oligo incorporation
into the genome. As part of this work, we have shown that oligos
can be used to introduce multiple changes into the genome
simultaneously. With oligos that are 40 nt in length, there is a
central core of greater than 15 nt that is almost always
incorporated. Longer oligos would be expected to have a
correspondingly longer core of nucleotide incorporation. One
Figure 7. Comparison of retention of oligo N sequences with
(59P) and without phosphorothioate linkages at the 59 end.
Data are from Fig. 3 and 4A. Approximately the same difference is
observed in msh6F strains.
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factor that is clearly important for the incorporation of a given part
of an oligo is its distance from either end of the oligo. MMR
represents a strong barrier to oligo transformation, and approach-
es involving transient inactivation of MMR appear most promising
in circumventing oligo rejection by MMR [7,39]. Even in the
absence of MMR, however, in yeast only a small fraction of cells
are transformed by a given oligo [5,6], for reasons that are not
entirely clear. The mechanism of incorporation suggests that for a
given cell, there would only be a short window for transformation
in which the region of interest was being replicated and had a
single-stranded region accessible for oligo annealing. Although the
stability of the oligo to cellular exonucleases could be an issue in
efficiency, protection of the ends with phosphorothioate linkages
seems to introduce other problems, and has not led to noticeable
increases in transformation efficiency in our hands. Another
possibility would be that oligos were effectively being inactivated
by protein binding or transport from the nucleus. Understanding
the remaining causes of low transformation efficiency will be
important for any potential therapeutic uses.
Conceived and designed the experiments: GPR GFC. Performed the
experiments: GPR JBS. Analyzed the data: GPR GFC. Wrote the paper:
1. Yamamoto T, Moerschell RP, Wakem LP, Komar-Panicucci S, Sherman F
(1992) Strand-specificity in the transformation of yeast with synthetic
oligonucleotides. Genetics 131: 811–819.
2. Yamamoto T, Moerschell RP, Wakem LP, Ferguson D, Sherman F (1992)
Parameters affecting the frequencies of transformation and co- transformation
with synthetic oligonucleotides in yeast. Yeast 8: 935–948.
3. Moerschell RP, Tsunasawa S, Sherman F (1988) Transformation of yeast with
synthetic oligonucleotides. Proc Natl Acad Sci USA 85: 524–528.
4. Li XT, Costantino N, Lu LY, Liu DP, Watt RM, et al. (2003) Identification of
factors influencing strand bias in oligonucleotide-mediated recombination in
Escherichia coli. Nucleic Acids Res 31: 6674–6687.
5. Kow YW, Bao G, Reeves JW, Jinks-Robertson S, Crouse GF (2007)
Oligonucleotide transformation of yeast reveals mismatch repair complexes to
be differentially active on DNA replication strands. Proc Natl Acad Sci USA
6. Rodriguez GP, Romanova NV, Bao G, Rouf NC, Kow YW, et al. (2012)
Mismatch repair dependent mutagenesis in nondividing cells. Proc Natl Acad
Sci USA 109: 6153–6158.
7. Aarts M, te Riele H (2011) Progress and prospects: oligonucleotide-directed gene
modification in mouse embryonic stem cells: a route to therapeutic application.
Gene Ther 18: 213–219.
8. Li GM (2008) Mechanisms and functions of DNA mismatch repair. Cell Res 18:
9. Iyer RR, Pluciennik A, Burdett V, Modrich PL (2006) DNA mismatch repair:
functions and mechanisms. Chem Rev 106: 302–323.
10. Jiricny J (2006) The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol
11. Hombauer H, Srivatsan A, Putnam CD, Kolodner RD (2011) Mismatch repair,
but not heteroduplex rejection, is temporally coupled to DNA replication.
Science 334: 1713–1716.
12. Brachman EE, Kmiec EB (2003) Targeted nucleotide repair of cycl mutations in
Saccharomyces cerevisiae directed by modified single-stranded DNA oligonucleo-
tides. Genetics 163: 527–538.
13. Maguire KK, Kmiec EB (2007) Multiple roles for MSH2 in the repair of a
deletion mutation directed by modified single-stranded oligonucleotides. Gene
14. Costantino N, Court DL (2003) Enhanced levels of lambda red-mediated
recombinants in mismatch repair mutants. Proc Natl Acad Sci USA 100: 15748–
15. Aarts M, te Riele H (2010) Subtle gene modification in mouse ES cells: evidence
for incorporation of unmodified oligonucleotides without induction of DNA
damage. Nucleic Acids Res 38: 6956–6967.
16. Dekker M, Brouwers C, Aarts M, van der Torre J, de Vries S, et al. (2006)
Effective oligonucleotide-mediated gene disruption in ES cells lacking the
mismatch repair protein MSH3. Gene Ther 13: 686–694.
17. Hegele H, Wuepping M, Ref C, Kenner O, Kaufmann D (2008) Simultaneous
targeted exchange of two nucleotides by single-stranded oligonucleotides clusters
within a region of about fourteen nucleotides. BMC Mol Biol 9: 14.
18. Radecke S, Radecke F, Peter I, Schwarz K (2006) Physical incorporation of a
single-stranded oligodeoxynucleotide during targeted repair of a human
chromosomal locus. J Gene Med 8: 217–228.
19. Agarwal S, Gamper HB, Kmiec EB (2003) Nucleotide replacement at two sites
can be directed by modified single-stranded oligonucleotides in vitro and in vivo.
Biomol Eng 20: 7–20.
20. Williams T-M, Fabbri RM, Reeves JW, Crouse GF (2005) A new reversion assay
for measuring all possible base pair substitutions in Saccharomyces cerevisiae.
Genetics 170: 1423–1426.
21. Storici F, Lewis LK, Resnick MA (2001) In vivo site-directed mutagenesis using
oligonucleotides. Nat Biotechnol 19: 773–776.
22. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, et al. (1999)
Functional characterization of the S. cerevisiae genome by gene deletion and
parallel analysis. Science 285: 901–906.
23. Goldstein AL, McCusker JH (1999) Three new dominant drug resistance
cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15: 1541–1553.
24. Sherman F (2002) Getting started with yeast. Methods Enzymol 350: 3–41.
25. Balakrishnan L, Bambara RA (2011) Eukaryotic lagging strand DNA replication
employs a multi-pathway mechanism that protects genome integrity. J Biol
Chem 286: 6865–6870.
26. Chilkova O, Stenlund P, Isoz I, Stith CM, Grabowski P, et al. (2007) The
eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-
ends via separate mechanisms but have comparable processivity in the presence
of PCNA. Nucleic Acids Res 35: 6588–6597.
27. Wang TC (2005) Discontinuous or semi-discontinuous DNA replication in
Escherichia coli? BioEssays 27: 633–636.
28. Pavlov YI, Shcherbakova PV (2010) DNA polymerases at the eukaryotic fork-20
years later. Mutat Res 685: 45–53.
29. Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM, Kunkel TA (2008)
Division of labor at the eukaryotic replication fork. Mol Cell 30: 137–144.
30. Guga P, Koziolkiewicz M (2011) Phosphorothioate nucleotides and oligonucle-
otides - recent progress in synthesis and application. Chem Biodivers 8: 1642–
31. Zheng L, Shen B (2011) Okazaki fragment maturation: nucleases take centre
stage. J Mol Cell Biol 3: 23–30.
32. Burgers PM (2009) Polymerase dynamics at the eukaryotic DNA replication
fork. J Biol Chem 284: 4041–4045.
33. Skerra A (1992) Phosphorothioate primers improve the amplification of DNA
sequences by DNA polymerases with proofreading activity. Nucleic Acids Res
34. Olsen PA, Solhaug A, Booth JA, Gelazauskaite M, Krauss S (2009) Cellular
responses to targeted genomic sequence modification using single-stranded
oligonucleotides and zinc-finger nucleases. DNA Repair (Amst) 8: 298–308.
35. Bonner M, Kmiec EB (2009) DNA breakage associated with targeted gene
alteration directed by DNA oligonucleotides. Mutat Res 669: 85–94.
36. Papaioannou I, Disterer P, Owen JS (2009) Use of internally nuclease-protected
single-strand DNA oligonucleotides and silencing of the mismatch repair
protein, MSH2, enhances the replication of corrected cells following gene
editing. J Gene Med 11: 267–274.
37. Rios X, Briggs AW, Christodoulou D, Gorham JM, Seidman JG, et al. (2012)
Stable gene targeting in human cells using single-strand oligonucleotides with
modified bases. PLoS ONE 7: e36697.
38. Nick McElhinny SA, Kissling GE, Kunkel TA (2010) Differential correction of
lagging-strand replication errors made by DNA polymerases a and d. Proc Natl
Acad Sci U S A 107: 21070–21075.
39. Dekker M, de Vries S, Aarts M, Dekker R, Brouwers C, et al. (2011) Transient
suppression of MLH1 allows effective single-nucleotide substitution by single-
stranded DNA oligonucleotides. Mutat Res 715: 52–60.
PLOS ONE | www.plosone.org7August 2012 | Volume 7 | Issue 8 | e42905