nihms91341.pdf

Common Mitochondrial DNA Mutations generated through DNA-

mediated Charge Transport

Edward J. Merino

†

Molly L. Davis

, and

Jacqueline K. Barton

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA

91125

Abstract

Mutation sites that arise in human mitochondrial DNA as a result of oxidation by a rhodium

photooxidant have been identified. HeLa cells were incubated with [Rh(phi)

bpy]Cl

(phi = 9,10-

phenanthrenequinone diimine), an intercalating photooxidant, to allow the complex to enter the cell

and bind mitochondrial DNA. Photoexcitation of DNA-bound [Rh(phi)

bpy]

can promote the

oxidation of guanine from a distance through DNA-mediated charge transport. After two rounds of

photolysis and growth of cells incubated with the rhodium complex, DNA mutations in a portion of

the mitochondrial genome were assessed via manual sequencing. The mutational pattern is consistent

with dG to dT transversions in the repetitive guanine tracts. Significantly, the mutational pattern

found overlaps oxidative damage hot spots seen previously. These mutations are found within

conserved sequence block II, a critical regulatory element involved in DNA replication, and these

have been identified as sites of low oxidation potential to which oxidative damage is funneled. Based

upon this mutational analysis and its correspondence to sites of long range oxidative damage, we

infer a critical role for DNA charge transport in generating these mutations and, thus, in regulating

mitochondrial DNA replication under oxidative stress.

INTRODUCTION

To protect cells from oxidative damage, a variety of repair processes and pathways must be

activated (

1-3

). Mitochondria are particularly susceptible because there may be increased levels

of toxic oxidants that are byproducts of oxidative phosphorylation within the mitochondrion

(4,5). These oxygen species react with mitochondrial DNA and promote mutations (6). The

most well studied modification to DNA is the oxidation of deoxyguanosine to 8-

oxodeoxyguanosine (8-oxodG), but 23 other modified bases have been characterized (7). It

has been observed that the 8-oxodG lesion leads to dG to dT transversions (

8); base oxidation

can also direct insertions and deletions, especially at microsatellites (9,10).

Here we examine mutations generated in mitochondria using the one-electron photooxidant

[Rh(phi)

bpy]

(phi = 9,10-phenanthrenequinone diimine). This complex binds to DNA by

intercalation with little sequence-specificity, and, when photoexcited at high energy (313-325

nm), direct strand cleavage by the DNA-bound Rh occurs, marking the site of intercalator

binding. Irradiation at longer wavelength (

≥

350 nm), however, promotes the oxidation of

guanines to yield common irreversible products including 8-oxodG (11). The guanines

oxidized need not, however, be localized to the Rh binding site, but instead oxidative damage

We are grateful to the NIH (GM49216) for their financial support of this research including a minority postdoctoral fellowship to E. J.

*to whom correspondence should be addressed at jkbarton@caltech.edu Tel. 626-395-6075. Fax 626-577-4976.

†

Present address: Department of Chemistry, University of Cincinnati, Cincinnati, OH, 45221-0172

NIH Public Access

Author Manuscript

Biochemistry

. Author manuscript; available in PMC 2010 February 3.

Published in final edited form as:

Biochemistry

. 2009 February 3; 48(4): 660–666. doi:10.1021/bi801570j.

NIH-PA Author Manuscript

to guanine doublets and triplets, sites of low oxidation potential in DNA, can be promoted from

a distance through DNA-mediated charge transport. When the Rh complex is tethered to the

end of a synthetic DNA oligonucleotide, oxidative damage has been observed ~200 Å from

the site of Rh intercalation (

12,13

). Indeed, varied photooxidants have been found to react with

DNA to promote oxidative damage to guanines from a distance (14). Holes injected into the

DNA base pair stack can migrate through the DNA base stack to low oxidation potential sites,

like the 5’-G of guanine doublets, to form guanine radicals that react with oxygen and water

to form permanent DNA lesions that require repair (15,16).

Figure 1 illustrates the strategy we have utilized to examine mutations derived through

oxidative damage. To promote oxidative stress in cells and specifically oxidative damage to

DNA, we incubate cells with [Rh(phi)

bpy]

and irradiate. Under these oxidative conditions,

some cells die, others undergo mutation, and many are left undamaged. Several rounds of Rh

incubation, irradiation, and regrowth can, however, lead to enrichment of the cell population

in mutants. These mutations can then be assayed by sequencing. It should be noted that the tris

(chelate) metal complexes are readily taken up inside cells, with the more lipophilic cationic

complexes taken up most efficiently (17). Rh complexes have been utilized to promote 1-

electron DNA oxidation with photoactivation in a variety of cell studies (16,18,19).

Most studies have shown that mutations within the mitochondria of tumors are not randomly

distributed but occur predominately in a sequence of the genome termed the control region or

d-loop (

20). Nucleotides 303-315 within the control region (sequence 3’-G

) are especially

prone to insertions and deletions as well as dG to dT mutations (

21). Nucleotides 303-315 are

termed conserved sequence block II, a regulatory element that is involved in primer formation

for mitochondrial DNA replication (22,23). Importantly, base oxidation arising from DNA

charge transport has been shown both

in vitro

and in HeLa cells to damage predominately this

conserved sequence block II (24,25). We hypothesize that, through DNA charge transport,

damage is funneled to the regulatory element, so as to hinder replication of a damaged

mitochondrion genome. While oxidative damage within conserved sequence block II,

generated through DNA charge transport, has been documented, here we shown that this

damage can lead to mitochondrial mutations. Moreover, we show that these mutations overlap

with known mutations associated with cancers.

EXPERIMENTAL

Methods

All experiments were carried out using newly thawed HeLa cells. HeLa cells (Sigma, USA)

were raised to ~75% confluence. Total DNA was purified using the DNAeasy kit (Qiagen,

U.S.A.) and eluted in 2.5 mM Tris, pH 8.5. PCR was used to amplify a 1.5 kb DNA

oligonucleotide that contained the control region using appropriate primers (24). Correct

amplification was verified using restriction digest.

Damage to a PCR product with [Rh(phi)

bpy]

Damage to a PCR product containing the control region by [Rh(phi)

bpy]

was accomplished

using 50 ng/

L of DNA in 50

L (5 mM Tris, 25 mM NaCl, pH 8) and addition of various

concentrations of [Rh(phi)

bpy]Cl

. Samples were irradiated at 365 nm for 15 min with a 1000

W Hg/Xe lamp outfitted with a 320 long-pass filter, then incubated with 99% piperidine [5

L, 10 min, 95 °C], cooled, and 5

L glacial acetic acid was added. The sample was prepared

for primer extension by purification with a PCR purification kit (Qiaquick, Qiagen) to give 40

L samples. A portion (30

L) of the sample was then mixed with 2X reaction mix [2X Taq

Buffer, 4 mM MgCl

, 200

M dNTP, 0.25

P-endlabelled primer, and 0.3 U/

L Platinum

Taq]. The sample was heated to 95 °C for 2.5 min. and cycled 40 times for primer extension

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[55 °C for 40 sec, 72 °C for 40 sec, 95 °C for 30 sec]. Samples were precipitated, and 6%

denaturing PAGE was performed. Gels were visualized by overnight exposure to a

phosphorimager screen and scanned. Data were quantified using the line function in

ImageQuant (Amersham) and normalized in Excel.

Damage to DNA in HeLa cells to generate mutations with [Rh(phi)

bpy]

Fresh HeLa cells were raised to ~75% confluence, 2 million total cells. Cells were trypsinized,

washed once with minimal media, pelleted, then resuspended in cold PBS and plated. Various

[Rh(phi)

bpy]Cl

concentrations in 2 mL PBS were added to the cells that were adherent to

the bottom of a 75 mL (25 cm

surface area) cell culture flask. Cells were irradiated at 37 °C

for 20 minutes with a 1000 W Orion Solar Simulator (with a UVB/UVC blocking filter and a

glass filter that eliminates wavelengths <345 nm) for 20 minutes. The Rh complex was then

removed by washing with PBS, and 50,000 cells were plated and allowed to grow. The

procedure was repeated four days later when cells had reached ~50% confluence. Following

the second round of photolysis, cells were again allowed to grow to ~50% confluence, and

total DNA was purified using the DNeasy kit (Qiagen, U.S.A.) and eluted from the column in

2.5 mM Tris, pH 8.5. The DNA was amplified by the addition of 20

L of 2X reaction mix

[2X Taq buffer, 4 mM MgCl

, 400

M dNTP, 1

M primer, and 10 U Taq] to 20

L each DNA

sample. The samples were heated to 95 °C for 2.5 minutes and cycled 40 times for primer

extension [55 °C for 40 seconds, 72 °C for 40 seconds, 95 °C for 30 seconds]. After purification

with the PCR Purification Kit (Qiagen, USA), sequencing reactions were accomplished by

addition of a terminator dNTP [1X Vent Buffer, 400

M dNTP, 150

M acyclonucleotide

(New England Biolabs), 5

P-endlabelled primer, 10 U Vent(exo-)] and cycled as listed

above. Samples were precipitated and electrophoresed on a 6% polyacrylamide gel. Gels were

visualized by overnight exposure to a phosphorimager screen and scanned. To visualize DNA

damage in an agarose gel, DNA was collected as above, loading dye was added (20% glycerol,

bromophenol blue, 1/10 volume), and electrophoresis was accomplished using ethiduim

bromide under standard conditions. Fluorescence was captured by a digital camera outfitted

with a uv-blocking filter and saved as tiff files. These files were quantified using Imagequant

as previously described (24,25). All experiments have been carried out at least in duplicate.

RESULTS

[Rh(phi)

bpy]

-induced oxidative DNA damage

Experiments to identify oxidative damage sites near conserved sequence block II were

conducted. Total DNA was collected from HeLa cells and amplified by PCR to give a 1.5

kilobase product that corresponds to the control region in human mitochondria. Primer

extension was carried out using a primer that begins at nucleotide 191 of the mitochondrial

genome, allowing for visualization of conserved sequence block II (

24). It should be noted that

extension of a PCR copy of the control region leads to a slightly different set of primer extension

products than with mitochondrial DNA purified from cells, examined previously (

25). Notably,

no primer extension stops are visualized within conserved sequence block II (Figure 2). These

extension stops in purified mitochondrial DNA are replication intermediates (26), triplex in

nature, and are removed during PCR. Primer extension from the control region PCR product

yields ~7-fold higher signal.

Sites of oxidation on the control region PCR product were identified using 2.5

g samples to

which 10 or 2

M [Rh(phi)

bpy]

was added. Irradiation at ~365 nm in the presence of DNA-

bound Rh leads to base oxidation, which can be revealed as a strand break by treatment of the

DNA with piperidine (

11,12

); after purification, a primer extension reaction is used to visualize

the DNA damage pattern. Irradiation at higher energy in the presence of Rh but no piperidine

treatment marks sites of Rh binding through direct strand cleavage. Sequencing is then used

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to identify the location of damage. With high energy irradiation and no piperidine treatment,

low level damage is obtained across the DNA, reflecting non-specific binding of the Rh

intercalator, and consistent with that seen earlier on mitochondrial DNA irradiated with Rh or

after irradiation of mitochondria in the presence of Rh (24,25). Addition of [Rh(phi)

bpy]

without irradiation or irradiation of the DNA without [Rh(phi)

bpy]

and piperidine treatment

shows a large, slowly migrating band that corresponds to the full length primer extension

product since no base oxidation occurs (Figure 2). Addition of [Rh(phi)

bpy]

and irradiation

leads to a series of stops at locations of base oxidation. With piperidine treatment, the strongest

stop is located at nucleotides 303-315, reflecting base oxidation at this site (Figure 2). These

data are fully consistent with previous studies on mitochondrial DNA purified from cells that

indicate conserved sequence block II is a particularly low oxidation potential site within the

mitochondrial genome (24,25). Though Rh binding is evident across the DNA (based upon

higher energy direct strand cleavage), base oxidation promoted by Rh is evident preferentially

in conserved sequence block II. Oxidative damage may thus be funneled to this site by DNA

charge transport. Position 263 is also observed as a low oxidation potential site and has been

previously identified as a site in purified mitochondrial DNA to which damage may also be

funneled (25). Damage at positions 286-291 was identified previously as a site that is

preferentially reactive to various chemical modifications (although direct strand cleavage with

Rh is not observed) (

24). Some Rh-specific damage is evident also at positions above 340, but

the exact locations have not been sequenced.

Assessing mitochondrial DNA damage in HeLa cells by [Rh(phi)

bpy]

HeLa cells (approximately 2×10

in each cell culture flask) were grown to confluence, dosed

with various concentrations of [Rh(phi)

bpy]

for 1 h, and then irradiated. After irradiation,

total DNA was collected from 1×10

cells and visualized on a 0.75% agarose gel stained with

ethidium bromide. As illustrated in Figure 3, when oxidation occurs, DNA is fragmented into

smaller random sizes. These smaller DNA fragments are below the detection limit of ethidium

bromide staining and, therefore, only a loss in the slow-moving, undamaged DNA band can

be observed as an indicator of the extent of oxidative damage in the HeLa cells. A 20 minute

irradiation at ~365 nm with no Rh added leads to less than 5% change in the DNA band. At 7

M [Rh(phi)

bpy]

, the high molecular weight DNA band loses 36% of its intensity, while

at 30

M Rh, 75% is lost. Therefore, extensive DNA damage occurs with incubation of [Rh

(phi)

bpy]

and irradiation.

The viability of cells after [Rh(phi)

bpy]

-induced oxidation was also examined. From the

samples above, 50,000 cells were plated and grown for an additional day. Cells irradiated

without Rh or with 7

M Rh yielded significant numbers of adherent cells, and new cells could

be seen in the microscope. In contrast, treatment with 30

M Rh and irradiation showed limited

numbers of adherent cells, ruptured cellular debris, and few new cells visible. DNA from the

adherent cells in all three cases was extracted, the control region of mitochondrial DNA was

amplified using PCR, and the product also visualized by agarose gel electrophoresis (Figure

3). With 30

M Rh irradiation, no DNA was detectable with PCR amplification. We therefore

utilized 7

M Rh and 20 minutes of irradiation in subsequent experiments; limited cell growth

occurred after too much DNA damage.

Inducing mitochondrial DNA mutations in HeLa cells

HeLa cells from a single thawed stock were then grown in three cell culture flasks. Once 50%

confluence was reached, cells from each flask were split into 50,000 and one million cell

samples. The one million cells were used for sequencing by extracting total DNA, PCR-

amplifying the control region, and nucleotides surrounding conserved sequence block II were

sequenced using an appropriate

P-labeled primer. The sequencing results are labeled as 0 in

Figure 4. Each set of 50,000 cells were plated and allowed to grow to 50% confluence. In one

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cell culture flask, 7

M Rh was added but not irradiated, the second flask was irradiated with

~365 nm light but no Rh, and, in the third flask, 7

M Rh was added and the flask was irradiated

to promote DNA damage. Each flask was then split into two flasks by adding 50,000 cells to

fresh media and grown to 50% confluence. One flask for each sample was used for sequencing

(this set is labeled 1 in Figure 4), while a second flask of cells was used to repeat the experiment

(labeled 2 in Figure 4, i.e. these cells have been oxidized twice). An important consideration

in these experiments is the number of cells to be plated after Rh-induced DNA damage. A

condition with too many cells leads to mutants not present in a high enough percentage for

definitive assignment, while with too few cells, the chance of finding a mutant is low.

Figure 4 shows line plots of guanosine sequencing results for nucleotides 284-330 from DNA

amplified with or without oxidative damage. These plots show conserved sequence block II to

the left and a nearby guanine 285 to the right for comparison (Figure 4). As expected,

sequencing line plots overlap when nothing has been done to the HeLa cells (labeled 0 in Figure

4), as these HeLa cells are genetically identical. Cells that were irradiated without Rh show no

changes in either position 285 or in positions 303-315, conserved sequence block II, after one

and two irradiations/growth cycles. In contrast, cells treated with [Rh(phi)

bpy]

and

irradiated show a decrease in guanine content within conserved sequence block II after two

rounds of oxidation and growth (compare plots on Figure 4 bottom after 2 cycles). Importantly,

position 285 shows little if any decrease after 0, 1, or 2 cycles of oxidation and growth,

indicating that the decrease in guanosine content is localized to the oxidation-prone regions to

which damage is funneled and does not reflect a global decrease in DNA yield. Analyzing

guanosine content at various positions within conserved sequence block II gives a variable

decrease in guanine content from as much a 42% to as little as 9% measured from peak heights.

Cells given Rh but not irradiated show a much smaller decrease in the guanosine content,

presumably, from ambient light absorbed during workup. The change with Rh addition and

irradiation is therefore significant. Mutations in oxidation hot spots are detectable after two

cycles of oxidation and growth.

Determining Specific Mitochondrial DNA Mutations

Another set of fresh cells was examined to identify the specific types of mutations that arise

upon Rh-induced DNA damage (Figure 5). Sequencing using the four terminating nucleotides

uncovers all types of mutation that occur. Cells were either treated with [Rh(phi)

bpy]

and

irradiated or treated with Rh without light as a control. Again, mutations become apparent after

two rounds of [Rh(phi)

bpy]

incubation and irradiation, while no mutations are seen with

cells not irradiated but treated with Rh (no DNA damage, Figure 5). Note that the primer is the

complement of the strand being interrogated. When visualizing 175 nucleotides of the control

region, mutations are evident at only four sites. The locations of mutations found are at positions

303-315, 286-291, 263, and 208-220. Interestingly, all positions have been identified as

nucleotides susceptible to Rh-induced DNA damage (24,25 and Figure 2).

In particular, both positions 303-315 and 263 are prone to oxidative damage via DNA charge

transport. Here, positions 303-315 show evidence of dG to dT transversions, seen as an increase

of 28% in dA content calculated from integration in the amount of dA over the 12 bases. Notably

the increase in dA content is specific to cells that have been through two rounds of Rh-induced

DNA damage and growth. A corresponding loss of dC is also found, although more blurry,

and as we would expect with either dG to dT transversions or deletions of dG (Figure 5). While

the dG to dT transversions are supported by the increase in dA content, deletions are also

suggested, since the increase in intensity (transversion mutations) observed in Figure 5B (green

plot) is uniform while the loss of dG (Figure 4) is non-uniform. These observations would be

consistent with deletions along with dG to dT transversions within conserved sequence block

II. Changes are also evident at position 263, consistent with mutation from a dG to a dT. We

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also found mutations at nucleotides 286-291 and 208-220, which are flexible sequences (24)

of T

and (AATT)

and are highly susceptible to mutation, particularly with irradiation. We

find at position 286-291 deletions or mutation from a dT to a dG (green arrow, Figure 5), while

positions 208-220 are mutated from a dA to a dT. These positions are damaged by a mechanism

that is not associated with DNA oxidation. Importantly, nucleotides 263 and 303-315 are

known as mutational hot spots in cancers (

24). Oxidative DNA damage through DNA charge

transport thus appears to lead to mutations that are characteristically found in tumors.

DISCUSSION

Hot spots for oxidative damage are present within the control region of the mitochondrial

genome and specifically within the highly conserved sequence block II. We have seen earlier

that damage is preferentially funneled to this region through DNA charge transport (24,25).

Here we see that long range damage in conserved sequence block II can also promote mutations.

Specifically, we find dG to dT transversions within the guanine-rich tracts of conserved

sequence block II. These mutations would be expected with oxidation of guanines to 8-oxodG;

other guanine oxidation products could also occur. In fact, we have identified 8-oxodG as one

product of long range oxidative damage in Rh-tethered oligonucleotides (

11,27). Importantly,

these lesions are commonly found associated with oxidative stress (5-7). Perhaps even more

interesting, however, is the role that DNA-mediated charge transport may play within the

mitochondrion. We observe that damage to this region of the mitochondrial genome and

therefore the mutations that occur as a result of this damage arise

from a distance

through DNA

charge transport. Indeed, we may consider now that DNA charge transport actually serves to

direct mutations to this region of the mitochondrial genome.

The preferential funneling of damage in the mitochondrion is seen to provide a checkpoint for

damage and oxidative stress within the cell. How that checkpoint may effectively operate or

become dysfunctional as a result of mutation is illustrated in Figure 6. Under oxidative stress,

damage to the mitochondrial genome occurs, and it is directed to conserved sequence block II

through DNA-mediated charge transport, leading to the formation of base lesions, like 8-

oxodG. Each mitochondrion has several mitochondrial DNA genomes, some of which may be

oxidatively modified while others remain undamaged. Though elements of mitochondrial DNA

replication remain unknown and controversial (22,23), replication will either be inhibited or

surely altered by DNA modification within the sequence block. We observe mutations

consistent with the formation of the 8-oxodG lesion within this region, but other oxidative

lesions associated with guanine oxidation have also been identified, and these too may inhibit

and/or alter replication (

28). Likely then, because conserved sequence block II represents both

the hot spot for damage and the replication control site, damage in this region should lead to

the selective replication of unmodified genomes.

Nonetheless, replication of damaged DNA can occur, albeit with low probability, and

replication of oxidized guanine lesions leads to mutation, specifically dG to dT transversions

and insertions/deletions within the guanine tract. The results described here identify these

specific mutations within conserved sequence block II and that these mutations arise through

the funneling of oxidative damage to these sites. Incubation of cells with [Rh(phi)

bpy]

, a

potent one-electron photooxidant that intercalates in DNA, with two cycles of irradiation and

cell growth, promotes specific mutation at the sites where Rh-induced damage on the

mitochondrial DNA has been identified.

Perhaps more remarkable is the finding that these specific mutations correspond to those

commonly associated with cancers. Such mutations certainly would hinder mitochondrial

function. Moreover, the loss of guanine content within the conserved sequence block would

mean that this region would no longer serve as a low oxidation potential checkpoint to limit

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replication of damaged genomes. Thus, mutations would further propogate, increasingly

limiting mitochondrial function. In fact, such mutations would be expected to provide an

advantage to tumor cells that utilize glycolysis preferentially for energy production. Therefore,

while mutation of conserved sequence block II itself may not be pathogenic, it may impart to

a tumor a higher chance to survive under hypoxic conditions.

These studies provide another example of how DNA-mediated charge transport may serve a

useful role within the cell (16). By funneling damage to specific control sites, the signal for

oxidative stress to the mitochondrial genome is transmitted. If replication proceeds despite this

signaling, mutations arise. Indeed, these mutations are characteristically associated with

cancerous transformations.

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Figure 1.

Strategy for inducing mutations in HeLa cell mitochondria. (A) Mitochondrial DNA under

elevated oxidative stress yields some oxidative damage to the mitochondrial genomes (red). If

unrepaired, modification of a dG leads to a mutation, here a dG to dT conversion. (B) In this

study HeLa cells are incubated with [Rh(phi)

bpy]

(bottom), a non-specific DNA intercalator

that, upon irradiation, induces DNA oxidative damage. Cells are either unmodified (blue),

modified (green and red), or dead (x on cell) after exposure to Rh and light. After growing the

cells, mutations can arise in the modified cells. Several cycles of oxidative damage and

regrowth lead to the accumulation of mutations that can be identified by sequencing.

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Figure 2.

[Rh(phi)

bpy]

-induced oxidative damage to the PCR-amplified mitochondrial DNA control

region. A 1.5 kilobase copy of the control region of mitochondrial DNA from HeLa cells was

prepared using PCR. After treatment with the [Rh(phi)

bpy]

photooxidant (10 or 2

M) and

incubation with piperidine, a primer extension reaction was used to visualize the DNA damage

via PAGE. Shown are sequencing lanes along with DNA treated with 10, 2

M, or 0 Rh with

or without irradiation as indicated. The nucleotide positions within the control region are shown

to the right. Addition of [Rh(phi)

bpy]

or irradiation alone do not produce oxidative damage

as only large, unmodified DNA is observed. Addition of [Rh(phi)

bpy]

, irradiation, and

piperidine treatment leads to oxidative damage at nucleotides 303, 290, and 263. The most

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intense damage is at position 303, within conserved sequence block II. Modification at

positions 303 and 263 are the result of DNA charge transport (red), damage at position 290 is

at a chemically accessible site of high AT content (green).

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Figure 3.

Agarose gel detection of global DNA damage in HeLa cells. Addition of [Rh(phi)

bpy]

HeLa cells and irradiation leads to DNA damage that is visualized as a loss of double-stranded,

high molecular weight DNA. Shown are amplified DNA samples after incubation of cells with

or without Rh (0, 7, 30

M) and irradiation for 20 min. The amount of DNA damage is sensitive

to the amount of photooxidant; 30

M [Rh(phi)

bpy]

causes more loss of unmodified DNA

than 7

M [Rh(phi)

bpy]

. Cells from these samples were grown for another 24 hours and

the mitochondrial control region was PCR-amplified. At 30

M [Rh(phi)

bpy]

, no

amplifiable DNA is seen.

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Figure 4.

Guanine sequencing of conserved sequence block II over several cycles of oxidation and

regrowth. Cellular DNA was extracted, the mitochondrial control region was amplified, and

sequencing was accomplished using a

P-labeled primer and dC terminator nucleotide. The

results shown are line plots of the PAGE gels from nucleotides 284-330. Conserved sequence

block II (303-315, 3’-G

, marked as CSB II) is a region comprised of many guanosine

residues and is prone to oxidation. A reference dG, that is not within conserved sequence block

II, at position 285 is on the right of each plot. At first, cells are genetically identical, and

sequencing lanes overlap (0). Cells were treated with [Rh(phi)

bpy]

only (no irradiation,

red), only irradiated with ~365 nm light (no Rh, blue), or given Rh and irradiated (Rh+light,

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black) and allowed to grow. Little, if any, change in the dG content occurs in conserved

sequence block II in all three cases after one cycle (labeled as 1). The same cells were treated

again (labeled as 2). After two cycles, cells treated with [Rh(phi)

bpy]

only (red) and only

irradiated (blue) have very similar dG content in conserved sequence block II. In contrast,

incubation of cells with [Rh(phi)

bpy]

and irradiation (black) shows a decrease in the dG

content in conserved sequence block II. The lack of change at position 285 with Rh and light

indicates the loss of dG content is specific to conserved sequence block II.

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Figure 5.

Mutations after Rh-induced DNA damage in HeLa cells. (A) Sequencing PAGE gel using all

four terminator nucleotides after two cycles of Rh-induced oxidative DNA damage and growth.

HeLa cells that were irradiated and treated with [Rh(phi)

bpy]

(DNA Damage plus) show

mutations observed as changes in the sequencing lanes (arrows) as compared to cells that where

incubated with Rh and not irradiated (DNA Damage minus). The mutations associated with

DNA charge transport (red) are consistent with conversion of dG to dT or deletion of dG. Other

mutations are also observed (green). (B) Line plot of the first two lanes of the same gel. Note

that it is the complementary strand that is sequenced, so that damage associated with a G to T

transversion yields an increase in the complementary A.

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Figure 6.

Schematic illustration how DNA charge transport may promote damage and mutations that

regulate mitochondrial replication. Mitochondria contain several genomes. Under oxidative

stress some genomes are damaged, and through DNA charge transport, this damage is

preferentially funneled to conserved sequence block II (green arrow). This funneling of damage

to conserved sequence block II can serve as a damage checkpoint, limiting the replication of

damaged mitochondrial genomes. Some damaged mitochondria can survive, however, and

here, mutations occur, as we have seen. These mutations may lead to improperly functioning

mitochondria and cancerous transformation.

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