Enhanced oligonucleotide-directed gene targeting in mammalian cells following treatment with DNA...

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Experimental Cell Researc

Enhanced oligonucleotide-directed gene targeting in mammalian cells

following treatment with DNA damaging agents

Luciana Ferraraa,b, Hetal Parekh-Olmedoa, Eric B. Kmieca,*

aDepartment of Biological Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, United StatesbDepartment of Genetics, Biology, Biochemistry, University of Torino, Italy

Received 14 April 2004, revised version received 22 June 2004

Available online 8 August 2004

Abstract

Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to

mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably

integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this

mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is

affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication

through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the

oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining

(NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is

repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces

or activates the homologous recombination pathway.

D 2004 Elsevier Inc. All rights reserved.

Keywords: DNA repair; DNA damage; Double-strand breaks; Single-stranded oligonucleotides; Nonhomologous end joining; Homologous recombination

Introduction

DNA oligonucleotides are being used to introduce single-

base changes into the genomes of prokaryotic and eukaryotic

cells [1]. The successful development of these kinds of

techniques will likely impact the fields of functional

genomics and gene therapy. While there are specific differ-

ences among the various approaches, including the strategies

used to deliver the oligonucleotide to the target cell or tissue,

the nucleotide alteration processes likely share common

features. It is currently believed that the reaction initializes

with a pairing phase in which the oligonucleotide is trans-

0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2004.06.021

* Corresponding author. Department of Biological Sciences, Delaware

Biotechnology Institute, University of Delaware, 15 Innovation Way,

Newark, DE 19711. Fax: +1 302 831 3427.

E-mail address: [email protected] (E.B. Kmiec).

ported to the nucleus and aligns in homologous register with a

complementary sequence in the genome. Once in alignment,

the complex attracts and engages endogenous DNA repair

systems that execute the base change. In prokaryotes, the

process of recombineering pioneered by Court et al. [2–4]

relies heavily on the activity of the E phage annealing protein

red h [5] to catalyze the pairing reaction. In eukaryotes,

triplex-forming oligonucleotides (TFOs) use nonenzymatic

activities to form the characteristic triple helix configuration

[6–8], while modified single-stranded oligonucleotides (non-

TFO) rely in all likelihood on recombinases such as Rad51 or

Rad52 [1,9–12].

While recombineering is achieving a remarkably high

level of nucleotide exchange (see Ref.[13] and references

therein), the frequency of gene repair in mammalian cells has

been unstable. There are numerous reasons for such

variability (see Ref. [1] and references therein), but one

h 300 (2004) 170–179

L. Ferrara et al. / Experimental Cell Research 300 (2004) 170–179 171

possibility is that the enzymes promoting the pairing or

repairing phases of the reaction are not present at sufficient

levels or in the required stoichiometry. Support for this notion

comes from recent data in which the overexpression of the

human RAD51 gene elevated the frequency of gene repair

only modestly (Hu et al., submitted for publication). In some

ways, this is not surprising because Rad51 participates in the

recombinational repair of DNA damage acting in conjunction

with a group of related proteins in a predetermined, optimal

stoichiometry [14]. In particular, this complex directs the

repair of double-stranded breaks that can occur during DNA

replication using the homologous recombination pathway in

some instances [15–19]. This response preserves and main-

tains genome stability.

Replicating DNA templates or at least cells in S phase

appears to be more amenable to alteration by single-stranded

oligonucleotides [20] for it is during this phase of the cell

cycle that the frequencies of gene repair are observed to be

higher [20] (Hu et al., submitted for publication). While

quiescent or nondividing cells contain a lower level of

proteins involved in HR and recombinational repair, cells in

S phase possess higher levels of these important proteins.

This relationship has recently been confirmed by studies

that detail correlations among cells in S phase, Rad51 foci

formation, and genetic recombination events [16,21,22].

Specifically, recombination between tandem repeats was

activated by replication arrest and the induction of double-

strand breaks (DSB) in a process known as recombination

induced by replication inhibition (RIRI) [22].

Because proteins involved in recombinational repair and

homologous recombination have been shown to increase the

frequency of gene repair in yeast [11,23], and DNA damage

stimulates homologous recombination pathways, we exam-

ined the effect of DNA damage on gene repair in

mammalian cells. Cells are grown under conditions where

the process of DNA replication is arrested or elongated and

double-strand breaks (DSB) are induced, the effects of

which supported a higher correction frequency. Our results

suggest that reducing the rate at which cells pass through S

phase, perhaps due to the accumulation of double-strand

breaks and the activation of the homologous recombination

pathway, leads to an improvement in the frequency of

targeted gene repair.

Materials and methods

Cell line and culture conditions

DLD-1 cells were obtained from American Type Cell

Culture (ATCC, Manassas, VA). DLD-1-integrated clone 1

(DLD-1-1) was obtained by integration of the vector

pEGFP-N3 containing a single point mutation (TAG) in

the eGFP gene. Cells were grown in RPMI 1640 medium

with 2 mM glutamine, 4.5 g/l glucose, 10 mM HEPES, 1

mM sodium pyruvate, and supplemented with 10% FBS.

Cells were maintained at 5% CO2, 378C and under selection

in 200 Ag/ml G418 (Gibco, Invitrogen Co., Carlsbad, CA).

eGFP gene targeting

Cells grown in complete medium supplemented with 10%

FBS were trypsinized and harvested by centrifugation. The

cell pellet was resuspended in serum-free medium at a density

of 1� 106 cells/100 Al and transferred to a 4 mm gap cuvette

(Fisher Scientific, Pittsburgh, PA). The oligonucleotide was

then added at a concentration of 4 AM and the cells were

electroporated (LV, 250 V, 13 ms, 2 pulses, 1 s interval) using

a BTX ECM830 apparatus (BTX, Holliston, MA). The cells

were then transferred to a 60-mm dish containing fresh

medium supplemented with 10% FBS and incubated for 48 h

at 378C before harvesting for FACS analysis.

Treatment of DLD-1 cell cultures with hydroxyurea, VP16,

or thymidine

Cells were seeded at a density of 0.8 � 106 cells 24 h

before addition of Hydroxyurea (HU) (1 mM), the eptop-

side, VP16 (3 AM), or thymidine (10 mM). The HU (Acros

Organics, Morris Plains, NJ) stock solution was prepared in

distilled water at a concentration of 500 mM and the VP16

(Sigma, St. Louis, MO) stock was prepared in DMSO

(100%) at a final concentration of 50 mM. Thymidine

(Sigma) stock was prepared in distilled water at a final

concentration of 200 mM. Hydroxyurea and VP16 were

added to the cells at the indicated concentration and time of

treatment was varied from 0–45 and 0–24 h, respectively.

Thymidine was added for a period of 24 h to synchronized

cells (see below).

Flow cytometry analysis

eGFP fluorescence of corrected cells was measured by a

Becton Dickinson FACS calibur flow cytometer (Becton

Dickinson, Rutherford, NJ). Cells were harvested 48 h after

electroporation and resuspended in FACS buffer (0.5%

BSA, 2 mM EDTA, 2 Ag/ml propidium iodide in PBS).

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 101

decade of the dot plot by adjusting the voltage for FL1

(GFP) and FL2 (PI). The 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 analyzed and those cells

being GFP positive and PI negative were scored as corrected

cells. For cell cycle analysis, 1 � 106 cells were plated 24 h

L. Ferrara et al. / Experimental Cell Research 300 (2004) 170–179172

before the treatment with drugs and after 24 h of treatment,

cells were trypsinized, resuspended in 300 Al cold PBS, and

fixed by adding 700 Al cold ethanol. Cells were then

resuspended in 1 ml of PBS containing 50 Ag/ml RNaseA

and 2.5 Ag/ml propidium iodide, and analyzed for DNA

content. The number of cells possessing actively replicating

forks was determined by BrdU staining (In Situ Cell

Proliferation Kit, FLUOS, Roche Diagnostics, Indianapolis,

IN) following manufacturers suggestions.

Pulsed-field gel electrophoresis

Twenty-four hours before treatment with HU orVP16, 1�106 cells were plated in tissue culture flasks, followed by

induction of DNA damage with HU or VP16 for 24 h. The

cells were released by trypsinization and melted in agarose

inserts. The agarose inserts were incubated in 0.5 M EDTA–

1% N-laurosylsarcosine-proteinase K (1 mg/ml) at 508C for

48 h and then washed four times in TE buffer before loading

on a 1% agarose gel (Pulse-Field Certified Agarose, Bio-Rad,

Hercules, CA) and DNA separation by pulsed-field gel

electrophoresis was carried out for 24 h (Bio-Rad, 1208 fieldangle, 60–240 s switch time, 4 V/cm). The gel was

subsequently stained with ethidium bromide and analyzed

Fig. 1. (A) DNA sequence of the target gene and the modified single-stranded oli

codon at position +67, is depicted with the (corrected) wild-type gene (lower pan

nonspecific (random) oligonucleotide (Hyg3S/74NT) are also shown. The asteri

represents modified phosphorothioate linkages. (B) FACS analysis of DLD-1 clon

(using propidium iodide staining). The control cells (left) were electroporated with

right represents mutant eGFP cells that were targeted with the specific oligonucleot

nonviable cells, while the lower right quadrant represents corrected eGFP (fluore

with AlphaImagerk 2200 (Alpha Innotech Corp., San

Leandro, CA).

Cell synchronization

Cells were synchronized in G1 or at the G1/S border by a

double thymidine block. Twenty-four hours before the

addition of any agent (HU, etc.), cells were plated at a

density of 0.5 � 106 cells per 100 mm dish, followed by

incubation in 2 mM thymidine (Sigma) for 16 h, washed and

released in fresh medium for 10 h, then incubated in 2 mM

thymidine for an additional 15 h. Cells were then washed

and either electroporated with oligonucleotide or treated

with HU (1 mM), VP16 (3 AM), or thymidine (10 mM) for

24 h before the electroporation step.

Results

Gene repair activity was assayed using a mutant eGFP

gene as a target. The wild-type gene was mutated at amino

acid 67 in the chromophore region so that no green

fluorescence is observed when it is expressed. The mutation

creates a stop codon (TAG) at a site that originally encoded a

gonucleotides used in this study. The mutant eGFP gene, containing a stop

el). The 72 base-specific oligonucleotide (EGFP3S/72NT) and the 74 base-

sk between the terminal of these bases in each oligonucleotide sequence

e-1-corrected cells. Cells were analyzed for eGFP expression and viability

a nonspecific oligonucleotide, Hyg3S/74NT, while the FACS graph on the

ide, EGFP3S/72NT (see text for details). The upper right quadrant represents

scent) cells.


tyrosine residue (TAC). We have previously used this mutant

gene as an episomal target for analyzing gene repair activity

[24], and Thorpe et al. [12] used a similar mutant eGFP target

to measure correction. For the present study, however, the

eGFP gene was integrated into DLD-1 cells using a pEGFP-

N3 vector generating a clonal cell line known as DLD-1-1

(clone-1) (Hu et al., submitted for publication). These cells

contain 2–4 copies of the mutant eGFP gene but do not

produce functional eGFP (see below). The DLD-1 cell line

was chosen because of its robust growth and its ability to

adapt to the transfer of oligonucleotides by square-wave

electroporation. This cell line has been reported to have a

deficiency in some aspects of homologous recombination

[25], but the cell line actually is heterogenous with several

subpopulations of cells [26], with varying responses to DNA

damaging agents [27]. To address this issue, we obtained

individual clonal isolates for our studies to avoid confusion

about its responsiveness to agents tested in the experiments

outlined in this paper.

The experimental strategy involved the introduction of

oligonucleotides into DLD-1-clone 1 cells by electroporation

followed by phenotypic readout of the corrected eGFP gene

48 h later. The targeting oligonucleotide is 72 bases in length

(72-mer), complementary to the nontranscribed strand of the

mutant eGFP gene but designed to create a single mismatch in

the third base of codon 67 (see Fig. 1). It directs conversion of

a TAGYTAC codon that enables phenotypic expression of

eGFP, which can be detected by FACS [see Ref. [24]. Fig. 1A

outlines the sequence of the target gene, the 72-mer, and a

nonspecific 74-mer used as a control. The time of addition of

certain agents such as hydroxyurea or VP16, relative to the

timing of electroporation of the oligonucleotide, is described

below (see Fig. 2).

Fig. 2. Both HU and VP16 stimulate gene repair in DLD-1 asynchronous cell cultu

added for 24 h to asynchronously growing cells. The percentage of green fluore

experimental point at various concentrations of either HU of VP16. (B) Cell vi

presented. Cell death was determined by propidium iodide staining and measured

each point are shown in the figure. CE, correction efficiency (*P value b 0.05; *

Fig. 1B demonstrates the usefulness and validity of the

eGFP system. Clone 1 cells were electroporated with either

EGFP3S/72NT or Hyg3S/74NT and the level of gene

correction measured 48 h later by FACS analysis. Approx-

imately 1.2% of the cells treated with EGFP3S/72NT score

positive for eGFP expression, but the frequency of correction

in any given experiment was observed to vary from 0.8% to

1.4%. No green fluorescence is observed in the control (lower

right quadrant) when the population of cells was treated with

the nonspecific oligonucleotide Hyg3S/74NT or with a

completely complementary oligonucleotide (data not

shown). As displayed in Fig. 1A, Hyg3S/74NT contains no

direct sequence complementarity to the mutant eGFP target

site.

Hydroxyurea and VP16 stimulate gene repair activity

Hydroxyurea can be used to synchronize growing cells in

S phase by blocking or retarding the movement of the

replication fork [28,29]. The effect on replication leads to

double-strand breaks at or near the fork [15], both of which

are repaired primarily by homologous recombination (HR)

[16,18]. VP16 (etoposide) is an anti-cancer drug that induces

DNA double-strand breaks through specific inhibition of the

resealing activity of topoisomerase II [30]. It is still not clear

if VP16-induced breaks occur preferentially at replication

forks or at random sites, but both treatments have been shown

to induce HR pathways and elevate the frequency of HR as a

result of DNA damage [15,16,18,22,31–33]. Because HR in

mammalian cells is induced by DNA damage [18] and our

previous data implicate HR in the process of gene repair

[11,34], we tested the hypothesis that the addition of DNA

damaging agents to mammalian cell cultures influences the

res. (A) Hydroxyurea (0, 0.3, 1, 2, 5 mM) or VP16 (0, 0.5, 1, 3, 10 AM) was

scent cells is presented as a function of the 50,000 cells assayed for each

ability posttreatment with HU or VP16 at the indicated concentrations is

by FACS analysis. Means and standard deviations for four experiments for

*P value b 0.01).

Fig. 3. Treatment with HU and VP16 increases the correction efficiency as a

function of exposure time to the drug. Cells were plated 24 h before adding

the addition of (A) HU (1 mM) or (B) VP16 (3 AM) for the time indicated.

Correction efficiency was determined as a percentage of eGFP-positive

cells after analyzing 50,000 cells. The means and standard deviations of two

to four experiments are shown (*P value b 0.05; **P value b 0.01).


process of gene repair. DLD-1 cells were incubated with

varying concentrations of hydroxyurea or VP16 and the level

of eGFP expression, a phenotypic measure of gene repair,

was analyzed. Cells were plated for 24 h before the addition

of HU or VP16 followed by electroporation 24 h later, and the

percentage of corrected cells was determined by FACS. As

shown in Fig. 2A, HU stimulated gene repair activity at each

concentration with a statistically significant difference

observed at 1, 2, and 5 mM, respectively. Similar results

were found in the VP16 experiments; stimulation is observed

at each concentration with statistically significant differences

observed at 1 and 3AM, respectively. VP16 elevates the gene

repair frequency up to 5% while the highest frequency

observed with HU treatment is 2.2%. The concentrations of

HU and VP16 that stimulate gene repair activity fall within

the range of concentration known to induce double-strand

breaks in mammalian cells [15,16,22].

The normal FACS assay used to detect gene repair is also

designed to measure cell death through the uptake of

propidium iodide and Fig. 2B displays cell survival after

treatment with increasing concentrations of HU or VP16.

While a modest amount of cell death is observed in cultures

treated with either agent, viability is slightly reduced at

higher concentrations, as observed previously [35–41].

We next evaluated enhancement of the frequency of gene

repair as a function of the time of exposure to either HU or

VP16. We incubated cells with 1 mM HU or 3 AM VP16 for

various times before the introduction of the oligonucleotide

under conditions where the overall time for each treatment

was the same but the time of exposure was varied. For

example, in the case of the 6 versus 24 h (VP16 treatment),

two cultures were grown for equal time (24 h), but in this

case the cells were treated with VP16 for the entire 24 h

while in the other case the cells were treated with VP16 for

the final 6 h. As shown in Figs. 3A and B, a gradual rise in

correction efficiency is observed up through 30–35 h with

HU and 12 h of exposure with VP16, respectively. The

asterisks in the figure indicate points that exhibit a statisti-

cally significant difference from the (zero) control: one

asterisk (*) = P value b 0.05; whereas two asterisks (**) = P

value b 0.01. These results indicate that longer contact time

with either agent leads to elevated frequencies of gene

repair, and using the terminology of Saintigny et al. [18], the

increase in gene repair activity can be classified as a blateresponseQ. Such a late response to double-strand DNA

breaks is catalyzed in large part by homologous recombi-

nation more so than nonhomologous end joining (NHEJ).

The concentration range of HU and VP16 used in our

experiments has been reported previously to induce DNA

damage, most often double-stranded DNA breaks [16,22].

But these conclusions were drawn from experiments con-

ducted in other cell lines, not the DLD-1 line. Thus, we

monitored the formation or accumulation of double-strand

breaks in DLD-1 cells by pulse-field gel electrophoresis

(PFGE) to assess the degree of DNA damage resulting from

the addition of HU or VP16. Cultures of asynchronously

growing DLD-1 cells were incubated with varying concen-

trations of HU or VP16 for 24 h and DNA breakage was then

assessed by PFGE. As shown in Fig. 4, a progressive increase

in the amount of damaged DNA, as a function of HU or VP16

concentration, is observed. Importantly, double-strand breaks

are found at the concentration of HU andVP16 that have been

shown coincidentally to stimulate the frequency of gene

repair. Our results are consistent with the observations of

Lundin et al. [16] and Saintigny et al. [18], and taken together

suggest a correlation between the two phenomena.

S phase has been observed to be the optimal period of the

cell cycle in which the highest levels of gene targeting are

obtained [20]. Besides inducing DNA damage, the addition

of HU or VP16 tomammalian cells in culture causes a stalling

of replication forks, as the cells respond to the DNA damage

and the metabolic stress. Because an enhancement of gene

repair activity is seen as a function of the addition of HU or

VP16 to growing cultures, it was of interest to determine what

percentage of the cells was actually in S phase at the time of

electroporation. The population of cells in this phase might

enable gene repair to take place more efficiently due to the

presence of actively replicating DNA forks and an overall

open chromatin structure. A modified nucleosomal config-

uration would likely be more accessible to the oligonucleo-

tide, which, in turn, would lead to higher levels of gene repair.

In this experiment, the cell cycle position of the treated and

Fig. 4. DSBs are induced in DLD-1 cells by exposure to HU and VP16, respectively. Double-strand breaks were visualized by PFGE after treatment with the

indicated concentrations of HU or VP16. Cells were treated for 24 h with HU (0.3, 1, 5 mM) or VP16 (0.5, 1, 3 AM) before collection and processing for gel

electrophoresis. Lane M indicates chromosomes of S. cerevisiae used as a marker, and lane C is a control of cells undergoing the same experimental

manipulation but without addition of HU or VP16.


untreated cells was determined by FACS after exposing cells

to VP16 or HU for 24 h. The results are shown in Fig. 5 (left

vertical column). While the profile of the untreated sample

depicts cells in all three phases, there is a substantial reduction

of cells in G2 as shown in the profiles of the HU or VP16 cell-

treated population. A coincident elevation in the percentage

of cells in S phase is also observed. These data are similar to

that obtained by Kumari et al. [42] in a similar series of

experiments measuring replication arrest associated with

double-strand breaks. Cells treated with VP16 also exhibit a

small peak of nonviable cells that represents a heightened

level of sensitivity of cells in S phase to this particular drug.

The percentage of cells in S phase was also determined by

BrdU staining in which BrdU becomes incorporated into

growing or replicating DNA strands. For each treatment

illustrated in FACS profile, the percentage of BrdU incorpo-

rating cells is presented in Fig. 5 (right column). The addition

of HU or VP16 to DLD-1 cells induces a higher percentage to

enter, remain, or stall in S phase. Our results correlate with the

cell cycle profiles obtained by Saintigny and Lopez [22] in

which a variety of replication inhibitors were tested for the

induction of HR activities. Thus, agents that induce DNA

damage simultaneously result in an accumulation of cells in S

phase; these two cellular responses may act synergistically to

elevate the frequency of gene repair.

Gene repair activity within a population of cells enriched

in S phase can be assessed directly by synchronizing the

cells using a double thymidine block followed by release

with subsequent electroporation of the targeting oligonu-

cleotide. If gene repair is more active on cells in S phase or

is elevated by a block in DNA replication incurred by

treatment with various agents, then a higher level of activity

would be predicted as more cells enter S phase together.

Alternatively, more cells could simply reside in S phase at

the time of the addition of the agent. The experiments

outlined in Fig. 6A were carried out by first synchronizing

the cells using a double thymidine block then releasing the

cells before electroporation. In the control reaction,

EGFP3S/72NT was electroporated at the time of release

and repair of the mutant eGFP gene was quantified 48 h

later by FACS. In some reactions, HU, VP16, or thymidine

was added to the culture at the time of release and EGFP3S/

72NT was electroporated after 24 h of treatment. HU and

VP16 had already been shown to enhance gene repair

activity when added for this amount of time to an

asynchronous population of DLD-1 cells (see Figs. 2 and

3). The effect of the double thymidine block on cell

synchrony is shown in Fig. 6B as the synchronized culture

exhibits a high percentage of cells in G1 or at the G1/S

border with concurrent reduction in the percentage of cells

in S phase (as judged by BrdU staining). The results of these

experiments indicate that the cell synchronization process

alone leads to a modest increase in the correction efficiency

as compared to asynchronously growing cultures (Fig. 6C).

The addition of HU stimulates gene repair activity

significantly in the synchronized culture raising the fre-

quency to greater than 9%. But, in contrast, the addition of

VP16 did not lead to a statistically significant difference in

the correction frequency of the cells in the synchronized

culture as compared to the asynchronously growing culture.

The single asterisk represents a statistically significant

difference between the synchronized and nonsynchronized

paired samples. We suggest that HU acts to stimulate gene

repair preferentially on a population of cells enriched in S

phase while VP16 exerts an equally stimulatory effect on

asynchronously or synchronously growing cultures.

Based on several criteria, including the temporal

response of gene repair observed in these studies, we

Fig. 5. The addition of HU or VP16 increases the percentage of cells present in S phase. Cells were treated with 1 mM HU or 3 AM VP16 for 24 h and then

analyzed by FACS for phasing in the cell cycle. The percentage of cells in S phase as a function of the addition of HU or VP16 was also determined by BrdU

incorporation. Samples treated with HU and VP16 were analyzed and compared to a sample that was not exposed to either agent. The total number of cells

incorporating BrdU and the relative percentage are presented in the right hand column.


hypothesized that hydroxyurea elevates the correction

activity by inducing HR primarily at replication forks. To

challenge this notion more directly, we utilized thymidine,

which is known to induce HR presumably by arresting cells

in S phase and acting to promote the creation of

recombination substrates within or at replication forks

[15]. Thymidine was substituted for HU in the reaction

described above and added to either an asynchronously

growing culture or a synchronized culture to a final

concentration of 10 mM. The oligonucleotide was electro-

porated 24 h after thymidine was added and gene repair

activity subsequently measured by FACS. Again, as shown

in Fig. 6C, the addition of thymidine stimulates gene repair

activity; elevating the correction efficiency approximately 8-

to 10-fold in the synchronized cell population. The

asynchronous culture exhibited only a modest response, if

any. Because more cells will contain replication forks in the

synchronized culture, under our experimental conditions,


this result is consistent with the notion that thymidine

produces substrates for HR at replication forks perhaps by

introducing damage or lesions in DNA templates actively

undergoing replication [15].

Discussion

Gene repair is a process in which a single base mutation

is altered within the context of the chromosome using

modified single-stranded oligonucleotides to direct the

reaction [see Ref. [1] for review]. The mechanism by which

the oligonucleotides act is not well understood, but the

pathway likely includes a DNA pairing step and a DNA

repairing phase [43]. Studies in yeast and mammalian cells

have helped identify candidate proteins that catalyze gene

repair, and some of these are known to function in

homologous recombination (HR) as well (Drury et al.,

submitted for publication) [11,34]. But proteins involved in

other pathways such as nonhomologous end joining (NHEJ)

could also participate in this novel reaction [44]. Consistent

with the notion that the HR pathway is active in gene repair

is the observation that overexpression of the human Rad51

recombinase elevates the frequency of gene repair both in

episomal [12] and chromosomal (Hu et al., submitted for

publication) systems. And now, Mre11, another protein

involved in HR, is active in the gene repair process [23].

Initially, we used hydroxyurea and VP16 to induce HR,

which act presumably by causing DNA damage [15,22,28–

30], and as judged by PFGE, both were found to induce

double-strand breaks in DLD-1 cells. The responses are

dose-dependent, and at higher concentrations, cell viability

is affected. Gene repair activity is seen to increase by

prolonged treatment with VP16 or HU, reaching a plateau at

or beyond 12 h, a time frame corresponding to the period

when activated or enhanced activity of a Rad51-dependent

pathway is observed [22].

DLD-1 cells, targeted for gene repair, accumulate in the

S phase as a function of treatment with either HU or

VP16. During this elongated phase, DNA lesions such as

double-strand breaks can occur, which by themselves may

stimulate DNA repair directed by proteins involved in the

HR pathway (recombinational repair). A current hypothesis

that defines the response to this type of DNA damage

centers on an early activation of the nonhomologous end

joining (NHEJ) pathway, which promotes cell viability,

followed by the activation of HR processes, which repair

Fig. 6. Correction efficiency is enhanced by cell synchronization followed

by treatment with HU or thymidine but not by VP16. (A) Outline of the

protocol for the cell synchronization study using a double thymidine block

to arrest the cells (see Materials and methods). After releasing the cells by

washing out the second thymidine block, the cells were incubated in the

presence of HU (1 mM), VP16 (3 AM), or thymidine (10 mM), respectively

for an additional 24 h. (B) Cell cycle analysis of synchronized cells after the

double thymidine block (t = 0) and at the time of treatment with HU

thymidine, or VP16 on electroporation. (C) The correction efficiency

(CE%) is depicted as a percentage of cells expressing eGFP from a total o

50,000 cells analyzed. The means and standard deviations from three o

four independent experiments for each point are shown (*P value b 0.05)

The asterisk indicates a statistically significant difference between the

synchronous and asynchronous cultures. Control, population of cells no

treated with any agent.

,

,

f

r

.

t


the subsequent (remaining) DNA damage [45–47]. Our

results do not directly address or exclude a role for NHEJ

in damage repair, but rather support a participatory role for

HR in gene repair. A previous report, however, did

indicate that HU activated both the nonhomologous and

the homologous recombination pathways, albeit to very

different levels; HR predominated in the overall response

[15]. But in other studies, double-strand breaks induced by

the action of the I-SceI enzyme in human glioma cells

were repaired predominantly by NHEJ between 24 and 48

h [48]. Thus, the dividing line, if one exists, between the

two pathways of chromosomal repair has not been made

concerning gene repair. The fact that the level of gene

repair continues to rise for a prolonged period after HU

addition, however, lends support to the involvement of

HR. Furthermore, the addition of thymidine to a popula-

tion of synchronized cells led to a stimulation of gene

repair activity and under these conditions, thymidine

induces homologous recombination exclusively [15].

Because we observe similar increases in correction

efficiency by the addition of either HU or thymidine, a

plausible conclusion is that gene repair activity is enhanced

when the HR pathway is induced.

Hydroxyurea appears to induce double-strand breaks

specifically at replication forks [15] while VP16 induces

breaks in a more random fashion [30]. Synchronized cells

treated with HU exhibit a higher level of gene repair while

synchronized cells treated with VP16 do not show increased

frequency beyond the levels obtained when VP16 is simply

added to asynchronously growing culture. The effect of HU

is best explained by the fact that more cells contain

replication forks when the culture is synchronized and

subsequently released into S, providing an increased number

of targets and accessibility for oligonucleotide-directed gene

repair activity. Because VP16 does not display a significant,

biased preference for replication forks, the level of

stimulation is predictably lower in synchronized cells

compared to that observed when HU is added.

DNA aberrations, lesions, or replication fork obstructions

result in the stalling of the replication process and cells

respond by extending their time in S phase where they

engage NHEJ or HR to help repair the damage. Coincident

with the repair of the structural damage in the DNA is an

increase in activity of gene repair, thus the results of our

study are consistent with an involvement of HR in the gene

repair reaction, at the very least as a bstimulatory agent.QThese conclusions are bolstered by new observations in

which the enhancement of gene repair activity induced by

the addition of VP16 was completely blocked when caffeine

was added to the reaction (Ferrara et al., in preparation).

Caffeine, a radiosensitizing agent, specifically inhibits

homologous recombination by blocking the activity of

ATM and ATR kinases, but it does not influence the process

of NHEJ [49,50]. In contrast, vanillin, an inhibitor of DNA-

PK activity in NHEJ, has no detectable effect on gene repair

reaction. Perhaps, the same complex of proteins, induced by

lesions accumulated during the process of DNA replication,

increases the activity of gene repair by its juxtaposition to

the site of DNA damage. Alternatively, the increase in HR

function may exhibit a more global effect on gene repair by

producing higher cellular concentrations of these critical

proteins. It is critical to note that our assay system does not

require a complete response of the HR pathway to generate

a positive signal. As mentioned above, certain clonal

isolates of DLD-1 cells may have differential HR activities

[25,27], but at present we have not yet determined if the

same proteins are required for both HR repair and gene

repair. What we can suggest is that stimulating the HR

response, as opposed to activating the NHEJ response, leads

to enhanced levels of gene repair. Elucidating the protein

participants will provide clues to the mechanism of this

interesting reaction that might eventually provide a basis for

utilizing gene repair as therapeutic.

Acknowledgments

This work was supported by grants from the National

Institutes of Health (R01 CA89325) and Tapestry

Pharmaceuticals.

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