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J Biol Chem. Mar 16, 2012; 287(12): 9222–9229.
Published online Dec 21, 2011. doi:  10.1074/jbc.M111.306852
PMCID: PMC3308766

Human Mitochondrial DNA Polymerase γ Exhibits Potential for Bypass and Mutagenesis at UV-induced Cyclobutane Thymine Dimers*An external file that holds a picture, illustration, etc.
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Abstract

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3′-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3′-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.

Keywords: DNA Damage, DNA Polymerase, DNA Replication, Mitochondrial DNA, Mutagenesis in Vitro, Ultraviolet Radiation

Introduction

Ultraviolet (UV) radiation is damaging to cellular macromolecules. DNA in particular is susceptible to several types of damage caused by UV irradiation; cyclobutane pyrimidine dimers and 6,4 photoproducts are the predominant types of damage caused by short wavelength, high energy UV radiation (1). Cyclobutane pyrimidine dimers have been shown to distort the DNA backbone only mildly, allowing almost normal Watson-Crick base-pairing at the affected site. Of the possible cyclobutane pyrimidine dimers formed, the cis-syn conformations predominate, and of these, thymine dimers (T-T)3 constitute about two-thirds (1). Because the effects of UV-induced thymine dimers are well characterized (28), UV radiation has been instrumental in the exploration of eukaryotic nuclear and bacterial DNA damage repair.

Unrepaired thymine dimers generally block synthesis of DNA by replicative polymerases (911), although limited capacity for bypass has been reported for some polymerases (12, 13). A complement of repair enzymes and translesion synthesis polymerases allow replication-blocking lesions to be repaired by nucleotide excision repair or bypassed in eukaryotic nuclear genomes as well as bacterial genomes (11, 12, 14, 15). Nuclear translesion bypass activity can be error-free or mutagenic, depending on the specific polymerase involved (16).

The mitochondrial genome, which is present in multiple copies in eukaryotic cells and replicated independently of the cell cycle (17, 18), is equally susceptible to UV-induced damage (19, 20). However, mitochondria lack nucleotide excision repair, so UV photoproducts are persistent in mitochondrial genomes (2123). Furthermore, mitochondria contain only one replicative DNA polymerase, DNA polymerase γ (18). How pol γ behaves when it encounters photodimers has never been tested.

The human pol γ holoenzyme consists of a catalytic subunit (encoded by POLG at chromosomal locus 15q25) and a dimeric form of its accessory subunit (encoded by POLG2 at chromosomal locus 17q24.1). The catalytic subunit is a 140-kDa enzyme (p140) that has DNA polymerase, 3′ → 5′ exonuclease, and 5′-deoxyribose phosphate lyase activities (18). The accessory subunit is a 55-kDa protein (p55) required for tight DNA binding and processive DNA synthesis (24).

We performed in vitro DNA polymerization reactions using a synthetic template oligonucleotide containing a single thymine dimer to measure the efficiency of incorporation and extension by pol γ opposite this lesion. Pol γ exhibited both accurate and mutagenic bypass. The efficiency of bypass was low compared with synthesis with an undamaged template but relatively high compared with other bulky lesions previously tested. Pol γ also showed higher efficiency of thymine dimer bypass than previously reported for other Family A polymerases (25). Our results suggest that pol γ most often stalls at thymine dimers, but it occasionally bypasses the lesion either accurately or aberrantly. These investigations offer a potential mechanism for short term tolerance of UV-induced mtDNA damage and also for long term UV-induced mtDNA mutagenesis.

EXPERIMENTAL PROCEDURES

Enzymes

A recombinant catalytic subunit of human DNA pol γ (exonuclease-proficient and exonuclease-deficient forms) containing a His6 affinity tag at its N terminus was overproduced in baculovirus-infected insect (Sf9) cells, and the protein was purified to homogeneity as described previously (2628). Two catalytic residues for exonuclease activity, Asp-198 and Glu-200, were substituted by alanines to construct the exonuclease-deficient pol γ, which abolishes all the 3′ → 5′ exonuclease activity of the enzyme (27). The p55 accessory subunit containing a His6 affinity tag at its C terminus was expressed in Escherichia coli and purified to homogeneity as described (24, 2729). The eluted protein samples were visualized with SDS-PAGE, and the proteins were frozen in small aliquots in liquid nitrogen and stored at −80 °C.

Substrates

Thymine dimer amidite was purchased from Glen Research and incorporated by TriLink BioTechnologies as the 17th-18th oligonucleotides from the 5′-end of the desired 45-mer template sequence 5′-CCAGCTCGGTACCGGGT-TAGCCTTTGGAGTCGACCTGCAGAAATT-3′. The oligonucleotide containing the thymine dimer was purified by reverse phase high pressure liquid chromatography, and homogeneity and purity were confirmed by mass spectrometry and polyacrylamide gel electrophoresis analysis, respectively, by TriLink BioTechnologies. A matching template with an undimerized thymine pair at the same site was purchased from Integrated DNA Technologies. Five primers for incorporation, and extension assays were purchased from Integrated DNA Technologies and labeled at their 5′-end with [γ-32P]ATP and T4 polynucleotide kinase. The sequences of these primers are 1) a 25-mer oligonucleotide, 5′-AATTTCTGCAGGTCGACTCCAAAGG-3′, 2) a 27-mer oligonucleotide, 5′-AATTTCTGCAGGTCGACTCCAAAGGCT-3′, 3) a 29-mer oligonucleotide containing two additional adenines at the 3′-end of the 27-mer primer, 4) a 28-mer oligonucleotide terminating with one additional adenine at the 3′-end of the 27-mer primer, and 5) a 28-mer oligonucleotide terminating with an additional guanine at the 3′-end of the 27-mer primer. The 5′-end-labeled primers were annealed to a 1.2-fold molar excess of each 45-mer template using standard protocols. Separate batches of the previously described 27- and 29-mer oligonucleotides containing a fluorescein covalently linked to their 5′-end were purchased from Sigma. These oligonucleotides served as primers in DNA binding reactions.

Single Nucleotide Incorporation/Extension Assays

Steady-state kinetic parameters were determined using polyacrylamide gel-based single nucleotide incorporation/extension assays. Reactions mixtures (10 μl) contained 25 mm HEPES-KOH (pH 7.5), 2 mm 2-mercaptoethanol, 0.1 mm EDTA, 5 mm MgCl2, 50 nm radiolabeled substrate, 10 nm exonuclease-deficient pol γ, and 20 nm p55. The reactions were started by the addition of one of the four dNTPs (at various concentrations depending on the substrate and analysis). After incubation at 37 °C for 10 min, reactions were terminated by the addition (10 μl) of 95% deionized formamide and 10 mm EDTA. Samples (3 μl) were boiled for 5 min at 95 °C and resolved by electrophoresis on 12% polyacrylamide gels containing 7 m urea and 25% deionized formamide. Gels were exposed to a phosphor screen, and the radioactive bands were detected with a Typhoon 9400 PhosphorImager (GE Healthcare) and quantified with NIH ImageJ software. Steady-state kinetic parameters, Km, and Vmax values were determined by fitting the data to the Michaelis-Menten model using KaleidaGraph (Version 4.1, Synergy).

Primer Extension Assays

Primer extension reactions were performed similar to the single nucleotide incorporation/extension assays but in the presence of all four dNTPs in the reaction mixture. The concentrations of dNTPs used for various extension analyses are summarized in Figs. 1 and and44.

FIGURE 1.
Replication bypass of thymine dimers catalyzed by human DNA pol γ. The substrates used for the assay (standing start, lanes 1–8 and 17–24, or running start, lanes 9–16 and 25–32) are represented schematically on ...
FIGURE 4.
Primer extension by exonuclease-deficient human DNA pol γ from various 3′-end terminated primers annealed to thymine dimer-containing templates. The substrate used for the assay is represented schematically on top of each autoradiogram ...

Exonuclease Assays

The rate of exonuclease activity was determined using a polyacrylamide gel-based assay. Reactions mixtures (150 μl) contained 25 mm HEPES-KOH (pH 7.5), 2 mm 2-mercaptoethanol, 0.1 mm EDTA, 50 nm radiolabeled substrate, 10 nm exonuclease-proficient pol γ, and 20 nm p55. Reactions were started by the addition of 5 mm MgCl2 at 37 °C and terminated by removing 10 μl of the mixture at different time points and adding it to tubes containing 10 μl of 95% deionized formamide and 10 mm EDTA. Gel electrophoresis and quantitation of radioactive bands were performed as described in the previous section. The rate of excision (kexo) of the 3′-primer terminus from each substrate was determined by plotting the loss of substrate against time (seconds) and fitting the data to a single-exponential using KaleidaGraph (Version 4.1, Synergy).

DNA Binding

Fluorescent substrates were prepared by hybridizing fluorescein-tagged primers to templates with or without T-T dimers. Immediately before use, the substrates were warmed to 37 °C for 5 min and held at room temperature. Steady-state fluorescence anisotropy was measured with an Olis RSM1000 spectrofluorometer (Bogart, GA) equipped with a 1.24-mm slit and a temperature-controlled cell set to 22 °C as previously described (30). Briefly, incident light at a 480-nm excitation wavelength was vertically plane-polarized and passed through a T-format quartz fluorometer cell, and fluorescence was measured at 2 identical detectors fitted with 520-nm high pass filters and vertical or horizontal polarizing filters. Binding mixtures (200 μl) contained 30 mm HEPES (pH 7.5), 1 mm 2-mercaptoethanol, 5 mm MgCl2, 0.01% Nonidet P-40, 50 mm NaCl, and 15 nm concentration of the specified fluorescein-conjugated oligonucleotide substrate. Changes in fluorescence polarization were measured in response to the stepwise addition of purified exonuclease-deficient pol γ. Anisotropy data were collected in triplicate with a 5-s integration time, after a 20-s equilibration period after each addition. Changes in anisotropy were plotted against the total concentration of protein. To correct for the ligand depletion effect caused by non-trivial concentrations of protein-DNA complex relative to the total protein concentration, binding isotherms were fit to a quadratic equation by non-linear regression analysis to calculate apparent Kd(DNA) values (31). Intrinsic fluorescence of buffer components was undetectable at wavelengths relevant to fluorescein.

RESULTS

DNA Pol γ Can Bypass Thymine Dimers with Reduced Efficiency

The ability of human pol γ holoenzyme to bypass T-T in template DNA was tested on two substrates, either a 5′-end-labeled 27-mer or a 25-mer primer annealed to a 45-mer template containing a single thymine dimer pair. The control substrates contained templates with undamaged thymines in an otherwise same sequence context. The resulting substrates contain two adjacent thymines either in dimeric or free form as the first two template bases (when annealed with the 27-mer primer) and as the 3rd and 4th bases (when annealed with the 25-mer primer) that pol γ will encounter after binding to the 3′-end of the primer terminus. Extension reactions were performed with both the exonuclease-deficient (Fig. 1, lanes 1–16) and exonuclease-proficient (Fig. 1, lanes 17–32) DNA pol γ holoenzyme as described in “Experimental Procedures” using various concentrations of dNTPs. The results suggested that the exonuclease-deficient DNA pol γ could bypass the T-T on the template and completely extend the primer strand with reduced efficiency compared with extension of an undamaged template with both substrates (Fig. 1, compare lanes 2–4, with lanes 6–8, and lanes 10–12 with lanes 14–16). The addition of an extra nucleotide at the end of the synthesis in the substrates containing the T-T dimer could be due to the 1000-fold higher concentrations of dNTPs used in these extension reactions compared with the control substrates (Fig. 1, Lanes 7, 8, 15, and 16). The exonuclease-proficient DNA pol γ barely bypassed the T-T dimer in the template even at high dNTP concentrations irrespective of the substrate (Fig. 1, compare lanes 18–20 with lanes 22–24 and lanes 26–28 with lanes 30–32).

Human Pol γ Preferentially Incorporates Purines Opposite T-T Dimers

The high concentration of dNTPs used by pol γ to bypass dimers in the template suggests that the enzyme might misincorporate nucleotides opposite this lesion. To investigate the nucleotide specificity of this apparent bypass event, single nucleotide incorporation assays were performed with the exonuclease-deficient pol γ using the same substrate described in the previous section, but this time only one of the four dNTPs was used in each reaction. The analysis revealed that pol γ could incorporate all four nucleotides opposite the undamaged thymine (Fig. 2, lanes 1–5). Although cognate base pairing was preferred based on steady-state kinetic analysis, pol γ incorporated guanine, cytosine, and thymine 200-, 10,100-, and 3,200-fold less efficiently compared with adenine opposite undamaged thymine in the template (Table 1 and supplemental Fig. 1). The incorporation of nucleotides opposite the thymine dimer was significantly reduced (Fig. 2, lanes 6–10). Pol γ incorporated two adenines opposite the T-T ~800-fold less efficiently compared with control (Table 1), primarily due to a ~650-fold reduction in Km for dATP binding (compare Km values of dA incorporation for ND and T-T-containing templates in Table 1). Surprisingly, guanine was incorporated only ~4-fold less efficiently than adenine opposite the 3′ but not 5′-thymine of the dimer (Fig. 2, compare lane 7 and 8; compare kcat/Km and relative efficiency (frel) for adenine versus guanine incorporation opposite T-T in Table 1), suggesting a possible role of pol γ in UV-induced mtDNA mutagenesis. Pol γ also incorporated pyrimidines opposite the 3′-thymine of the dimer but much less efficiently (Fig. 2, lanes 9 and 10; Table 1, kcat values of dC and dT incorporation opposite T-T in the template; supplemental Fig. 1).

FIGURE 2.
Single-nucleotide incorporation catalyzed by exonuclease-deficient DNA pol γ opposite the dimer-containing templates. Schematic representation of the substrate used with the location of the thymine dimer is shown on the top of the autoradiogram ...
TABLE 1
Steady-state kinetic parameters for nucleotide incorporation by exonuclease-deficient human pol γ opposite thymine dimers in DNA template

Exonuclease Activity of Pol γ Is Not Affected by T-T Dimer-containing Substrates

Because pol γ preferred to incorporate purines opposite to the thymine dimers, three possible primer termini could exist during the bypass event. The 3′-end of the primer termini could contain either two adenines opposite the T-T or an adenine or a guanine opposite the 3′-thymine of the dimer (See Table 2). To determine whether these three primer termini were more or less recognized and degraded by the exonuclease activity opposite T-T dimers compared with undamaged thymines in template, exonuclease assays were performed using exonuclease-proficient DNA pol γ. The results from the analysis revealed that the excision rates (kexo) of all three primer termini increased only 1.1- 2.8-fold for substrates containing the T-T compared with undamaged thymines, suggesting that the incorporated purines were only moderately selected for digestion opposite T-T dimers relative to undamaged thymines (Table 2).

TABLE 2
Excision rate of various 3′-primer terminus opposite to the thymine dimers by exonuclease-proficient human DNA pol γ

Rate-limiting Step in Replication Bypass Is translesion synthesis Opposite T-T Dimers

Next, we tested whether pol γ could perform extension past the T-T efficiently after incorporation of purines opposite the dimer. To determine the efficiency of extension past the three primer termini (mentioned in the previous section), single nucleotide extension assays were performed with exonuclease-deficient pol γ (Fig. 3 and Table 3). Steady-state kinetic analyses revealed that in the presence of two adenines opposite the dimer, extension past the lesion with cytosines was only 50-fold less efficient compared with the control substrate (Table 3 and supplemental Fig. 2). However, pol γ was 1,840-fold less efficient at incorporating an adenine opposite the 5′-thymine with an adenine already opposite the 3′-thymine of the damaged template compared with the undamaged template (Table 3). The catalytic efficiency of the polymerase also decreased 210-fold when incorporating an adenine opposite the 5′-thymine of the T-T after a misincorporated guanine opposite the 3′-thymine of the dimer when compared with non-damaged thymines in the template.

FIGURE 3.
Single-nucleotide extension analysis of exonuclease-deficient DNA pol γ past the thymine dimers in template. Schematic representation of the substrate used with the location of the thymine dimer is shown on the top of the autoradiogram with the ...
TABLE 3
Steady-state kinetic parameters for extension opposite to or past the thymine dimers by exonuclease-deficient human DNA pol γ

We tested the ability of the exonuclease-deficient human pol γ to perform primer extension on various 3′-end terminated primers annealed to dimer-containing templates. As suggested by our single nucleotide incorporation analysis and steady-state kinetics, pol γ could perform complete extension of all four primers with different efficiencies at various nucleotide concentrations (Fig. 4). An ~1000-fold excess of dNTPs was required to observe noticeable amounts of full-length products for all translesion synthesis past either one or two bases of thymine dimers in comparison to the control substrates (Fig. 4, compare lanes 2–4 to 6–8, lanes 18–20 to 22–24, and lanes 26–28 to 30–32). However, when the 3′-end of the primer termini contained two adenines annealed to the thymine dimers of the template strand, pol γ extended significant quantities of the primer to full-length products in the presence of only a 50-fold excess of nucleotides as compared with the reactions containing undamaged templates (Fig. 4, compare lanes 10–12 to 14–16). This result in addition to the primer extension and steady-state kinetic analysis suggested that the major rate-limiting step in bypassing the thymine dimer lesions in mtDNA is the incorporation of nucleotides opposite the dimers and not extension past the damaged thymine bases.

Thymine Dimer-containing Templates Do Not Affect Pol γ-DNA Interactions

Because translesion synthesis and bypass of T-T by pol γ requires binding to dimer containing primer-template substrates, we performed fluorescence anisotropy measurements to determine the binding affinities of the exonuclease-deficient pol γ to substrates containing thymine dimers in solution at equilibrium conditions. This method allowed us to measure the increase in fluorescence anisotropy, which is directly proportional to the binding of pol γ to the 3′-end of primer-template substrates, causing a decrease in rotational diffusion of the protein-DNA complex. The substrates used for this study contained primers (27- or 29-mer) with a covalently linked fluorescein at its 5′-end annealed to templates containing either a thymine dimer or two adjacent undamaged thymines in the same sequence context. The substrate containing the 27-mer primer would have exposed thymine dimers on the template strand, whereas in the substrate with 29-mer primer the T-T dimer would be annealed to two 3′-adenines of the primer (see Table 4). Substrates were mixed with increasing concentrations of the polymerase enzyme, and binding curves were generated by plotting changes in fluorescence anisotropy against total protein concentration. The apparent Kd(DNA) values were subsequently calculated by fitting a quadratic equation to the isotherms as described under “Experimental Procedures.” The results from the analysis revealed that the Kd(DNA) for pol γ ranged from 102 ± 10 to 143 ± 15 nm depending on the substrate used (Table 4), suggesting that the presence of a thymine dimer in the template did not significantly affect the ability of pol γ to bind to the DNA substrates used in this study, and hence, translesion synthesis and bypass of the dimer is not limited by kinetics of pol γ-DNA interactions.

TABLE 4
DNA binding affinity of exonuclease-deficient human DNA pol γ on various substrates containing thymine dimers

DISCUSSION

Mitochondrial DNA polymerase, pol γ, encounters various types of damage in the mitochondrial genome, but interactions between pol γ and lesions in the mitochondrial genome have not been well characterized. Pol γ stalls at abasic sites, and some bulky adducts such as polycyclic aromatic hydrocarbon and cisplatin adducts have been shown to block incorporation and extension by pol γ in vitro (3234). There is evidence for translesion synthesis by pol γ with other platinated adducts (34), and pol γ readily bypasses 8-oxo-dG lesions (32), although with reduced fidelity. Replication of mtDNA is hindered in vivo after dosing with UVC (35), and mtDNA mutations and deletions have been clinically linked to UV exposure (3639). However, the details of the interaction of pol γ with UV-induced thymine dimers have not previously been described.

In these experiments we investigated the ability of pol γ to fully extend a template containing a single T-T dimer in the presence of all four nucleotides and the specificity of individual nucleotide incorporation opposite and after the lesion. We also tested for full extension after several possible incorporation scenarios at the lesion site. We found that pol γ can fully extend a primer opposite a dimer-containing template, although more slowly than opposite an undamaged template. The 800-fold decrease in efficiency of incorporation of correct adenines at the damage site suggests that very few T-T-containing mitochondrial genomes would be replicated efficiently. Moreover, the exonuclease-proficient pol γ greatly reduced the translesion synthesis observed in Fig. 1 but did allow a small population to bypass. DNA pol γ was previously shown to have a preference for excising mismatched nucleotides to matched nucleotides from the 3′-end (40, 41). Although the human pol γ demonstrated a preference for excising the guanine opposite the undamaged thymine in template relative to excising an adenine (Table 2), no preference was noted for removal of guanine opposite T-T dimers relative to removal of adenine. The reduced product formed by the exonuclease-proficient pol γ in Fig. 1 (lanes 22–24 and 30–32) is most likely a result of the lower incorporation of the first nucleotide opposite the T-T dimer (Table 1). Thus, our results suggest that most of the replication complexes would stall, leaving the damaged genomes unreplicated. Depending on the frequency of such lesions and the mtDNA copy number, this could lead to mtDNA depletion. It is also possible that stalling could lead to double-strand breaks, which has been proposed as a mechanism for the introduction of mtDNA deletions (42). Of note, one report indicates that mitochondrial dNTP levels vary from ~3 μm for dATP to 140 μm for dGTP in different rat tissues (43), suggesting that the levels of dNTP we used to drive T-T bypass with pol γ were in the range found in rat tissue, and hence, bypass appears to be feasible. Furthermore, the ability of pol γ to bypass UV lesions may increase after DNA damage. In response to damage to DNA, p53 is activated, which then increases mitochondrial nucleotide pools by the induction of the p53 inducible small subunit of ribonucleotide reductase, RRM2B (44, 45). Nucleotide pools imported from the cytoplasm due to ribonucleotide reductase activity may serve as the main supply of mitochondrial nucleotide pools, where the salvage pathway serves as a backup (46). Thus, the up-regulation of nucleotide pools via the p53 induction after UV damage may allow more bypass of thymine dimers.

Long term effects might also be caused by replication of remaining mtDNAs that carry mutations, resulting over time in the percentage of mutated copies reaching the threshold required to cause a physiological effect. Although all (wild-type and mutant) mtDNAs in a population of heteroplasmic cells would suffer the same average level of damage, some cells will by chance contain more UV-damaged wild-type than mutant mtDNAs, which could lead to an enrichment of the mutant mtDNAs in those cells and their daughter cells. In addition, there is evidence that mitochondrial genomes carrying large deletions are preferentially replicated compared with normal genomes (47).

Furthermore, although bypass would be expected to occur infrequently based on our results, the large number of mtDNA replication events that occur in skin cells over an individual's lifetime combined with the thousands of copies of the genome present in each cell suggest that bypass will occasionally occur. This would be especially true in cells that have been exposed to moderate, non-cytotoxic levels of UV radiation and carry a significant load of T-T dimers in their mtDNA; this is a likely exposure scenario given the chronic and lifelong nature of sunlight exposure. In the event of bypass, pol γ is only four times less efficient at misincorporating a guanine opposite the 3′-thymine of the dimer than correctly incorporating adenine. Additionally, in the presence of all four dNTPs, pol γ can fully extend a primer containing a guanine opposite the 3′-thymine, indicating that misincorporation opposite the dimer will not stall further synthesis. This misincorporation in the absence of exonuclease activity would leave a point mutation, specifically an A → G transition, in its wake. There is some evidence for mismatch repair in mammalian mtDNA (48, 49), but this process is poorly understood.

Because mitochondria lack nucleotide excision repair needed for repairing T-T dimers, these disruptive lesions will persist in mtDNA (2123). In addition, mtDNA is replicated repeatedly during the lifetime of the cell and independently of the cell cycle, amplifying the impact of mutation-causing lesions. Human mtDNA contains very little non-coding sequence (17, 39), suggesting that mutations are more likely to alter or disrupt protein function. Imbalances in the levels of the 13 proteins encoded by most mitochondrial genomes might lead to mitochondrial dysfunction, reactive oxygen species production, and disruption of normal apoptotic regulation (50, 51). Additionally, the accumulation of point mutations in the non-coding control region of mtDNA, which helps control replication and transcription of the mitochondrial genome, has been associated with aging in several human tissues (38, 39).

A growing number of epidemiological studies point to correlations between mtDNA damage, UV exposure, and skin disease (3739, 52). Photoaging of the skin has been closely tied to mtDNA mutations and deletions (36, 53, 54), and there is recent evidence associating mtDNA damage with several types of skin cancer (5559). A handful of studies that have tested mtDNA from melanoma and non-melanoma skin cancer patients have revealed a number of A → G and T → C point mutations (56, 57, 59), often in the context of pyrimidine:pyrimidine dimers. These do not fit the signature UV point mutation found in nuclear DNA, a C → T substitution. However, based on our results, they do coincide with point mutations that could be generated by pol γ inaccurately bypassing a thymine dimer with a guanine. After the following round of replication, the A:T pair (with the thymine of the dimer) would be replaced by a G:C pair, which would be normally copied by pol γ for all the following replication events, introducing a permanent point mutation into the set of mtDNA copies generated from this daughter copy. However, it should be noted that additional mtDNA point mutations as well as deletions have also been observed in skin cancer, and our work cannot on its own provide insight into the relative importance of 6,4-photoproducts or other (e.g. oxidative) damage in ultraviolet light-mediated mtDNA mutagenesis.

Our results demonstrate that UV damage to mtDNA can disrupt normal, accurate replication by pol γ. The degree to which the disruption we have observed has a physiological effect on cells and tissues requires further investigation.

Supplementary Material

Supplemental Data:

Acknowledgments

We are thankful to Dr. Matthew J. Longley for assistance with fluorescence anisotropy experiments, Dr. Danielle L. Watt for help with troubleshooting our acrylamide gels, and Dr. Christal D. Sohl for technical advice on exonuclease assays. We also thank Drs. Jeffrey D. Stumpf and Matthew J. Young for critical comments on the manuscript.

*This work was supported, in whole or in part, by National Institutes of Health Grant R21 NS065468 (to J. N. M.) and ES 065078 (to W. C. C.).

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Figs. 1 and 2.

3The abbreviations used are:

T-T
thymine dimer
pol γ
polymerase γ
ND
non-damaged.

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