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Common Mitochondrial DNA Mutations Generated Through DNA-Mediated Charge Transport† Edward J

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Common Mitochondrial DNA Mutations Generated Through DNA-Mediated Charge Transport† Edward J Subscriber access provided by Caltech Library Services Article Common Mitochondrial DNA Mutations Generated through DNA-Mediated Charge Transport† Edward J. Merino, Molly L. Davis, and Jacqueline K. Barton Biochemistry, 2009, 48 (4), 660-666• DOI: 10.1021/bi801570j • Publication Date (Web): 07 January 2009 Downloaded from http://pubs.acs.org on March 13, 2009 More About This Article Additional resources and features associated with this article are available within the HTML version: • Supporting Information • Access to high resolution figures • Links to articles and content related to this article • Copyright permission to reproduce figures and/or text from this article Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Subscriber access provided by Caltech Library Services Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 660 Biochemistry 2009, 48, 660–666 Articles 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, California 91125 ReceiVed August 20, 2008; ReVised Manuscript ReceiVed October 31, 2008 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)2bpy]Cl3 (phi is 9,10- phenanthrenequinone diimine), an intercalating photooxidant, to allow the complex to enter the cell and 3+ bind mitochondrial DNA. Photoexcitation of DNA-bound [Rh(phi)2bpy] 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. On the basis of 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. To protect cells from oxidative damage, a variety of repair 8-oxodG (11). The guanines oxidized need not, however, processes and pathways must be activated (1-3). Mitochon- be localized to the Rh binding site; instead, oxidative damage dria are particularly susceptible because there may be to guanine doublets and triplets, sites of low oxidation increased levels of toxic oxidants that are byproducts of potential in DNA, can be promoted from a distance through oxidative phosphorylation within the mitochondrion (4, 5). DNA-mediated charge transport. When the Rh complex is These oxygen species react with mitochondrial DNA and tethered to the end of a synthetic DNA oligonucleotide, promote mutations (6). The most well-studied modification oxidative damage has been observed ∼200 Å from the site to DNA is the oxidation of deoxyguanosine to 8-oxodeox- of Rh intercalation (12, 13). Indeed, varied photooxidants yguanosine (8-oxodG), but 23 other modified bases have have been found to react with DNA to promote oxidative been characterized (7). It has been observed that the 8-oxodG damage to guanines from a distance (14). Holes injected into lesion leads to dG to dT transversions (8); base oxidation the DNA base pair stack can migrate through the DNA base can also direct insertions and deletions, especially at ′ microsatellites (9, 10). stack to low-oxidation potential sites, like the 5 -G of guanine doublets, to form guanine radicals that react with oxygen Here we examine mutations generated in mitochondria 3+ and water to form permanent DNA lesions that require using the one-electron photooxidant [Rh(phi)2bpy] (phi is 9,10-phenanthrenequinone diimine). This complex binds to repair (15, 16). DNA by intercalation with little sequence specificity, and Figure 1 illustrates the strategy we have utilized to examine when the complex is photoexcited at high energy (313-325 mutations derived through oxidative damage. To promote nm), direct strand cleavage by the DNA-bound Rh occurs, oxidative stress in cells and specifically oxidative damage 3+ marking the site of intercalator binding. Irradiation at longer to DNA, we incubate cells with [Rh(phi)2bpy] and irradiate. wavelengths (g350 nm), however, promotes the oxidation Under these oxidative conditions, some cells die, others of guanines to yield common irreversible products, including undergo mutation, and many are left undamaged. Several rounds of Rh incubation, irradiation, and regrowth can, † We are grateful to the NIH (GM49216) for their financial support however, lead to enrichment of the cell population in mutants. of this research, including a minority postdoctoral fellowship to E.J.M. These mutations can then be assayed by sequencing. It should * To whom correspondence should be addressed. E-mail: jkbarton@ be noted that the tris(chelate) metal complexes are readily caltech.edu. Telephone: (626) 395-6075. Fax: (626) 577-4976. ‡ Present address: Department of Chemistry, University of Cincinnati, taken up inside cells, with the more lipophilic cationic Cincinnati, OH 45221-0172. complexes taken up most efficiently (17). Rh complexes have 10.1021/bi801570j CCC: $40.75 2009 American Chemical Society Published on Web 01/07/2009 DNA Mutations Generated through DNA-Mediated Charge Transport Biochemistry, Vol. 48, No. 4, 2009 661 3+ Damage to a PCR Product with [Rh(phi)2bpy] . Damage to a PCR product containing the control region by 3+ [Rh(phi)2bpy] was accomplished using 50 ng/µL DNA in 50 µL [5 mM Tris and 25 mM NaCl (pH 8)] and addition of various concentrations of [Rh(phi)2bpy]Cl3. 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 µL, 10 min, 95 °C), and cooled, and 5 µLof glacial acetic acid was added. The sample was prepared for primer extension by purification with a PCR purification kit (Qiaquick, Qiagen) to give 40 µL samples. A portion (30 µL) of the sample was then mixed with 2× reaction mix 32 (2× Taq buffer, 4 mM MgCl2, 200 µM dNTP, 0.25 µM P- end-labeled primer, and 0.3 unit/µL platinum Taq). The sample was heated to 95 °C for 2.5 min and cycled 40 times for primer extension (55 °C for 40 s, 72 °C for 40 s, and 95 °C for 30 s). Samples were precipitated, and 6% denaturing FIGURE 1: Strategy for inducing mutations in HeLa cell mitochon- PAGE was performed. Gels were visualized by overnight dria. (A) Mitochondrial DNA under elevated oxidative stress yields exposure to a phosphorimager screen and scanned. Data were some oxidative damage to the mitochondrial genomes (red). If quantified using the line function in ImageQuant (Amersham) unrepaired, modification of a dG leads to a mutation, here a dG to and normalized in Excel. dT conversion. (B) In this study, HeLa cells were incubated with 3+ [Rh(phi)2bpy] (bottom), a nonspecific DNA intercalator that, upon Damage to DNA in HeLa Cells To Generate Mutations irradiation, induces DNA oxidative damage. Cells were either 3+ ∼ unmodified (blue), modified (green and red), or dead (x on the cell) with [Rh(phi)2bpy] . Fresh HeLa cells were raised to 75% after exposure to Rh and light. After the cells have been grown, confluency, 2 million total cells. Cells were trypsinized, mutations can arise in the modified cells. Several cycles of oxidative washed once with minimal medium, pelleted, resuspended damage and regrowth lead to the accumulation of mutations that in cold PBS, and plated. Various [Rh(phi)2bpy]Cl3 concen- can be identified by sequencing. trations in 2 mL of PBS were added to the cells that were adherent to the bottom of a 75 mL (25 cm2 surface area) been utilized to promote one-electron DNA oxidation with cell culture flask. Cells were irradiated at 37 °C for 20 min photoactivation in a variety of cell studies (16, 18, 19). with a 1000 W Orion Solar Simulator (with a UVB/UVC Most studies have shown that mutations within the blocking filter and a glass filter that eliminates wavelengths mitochondria of tumors are not randomly distributed but of <345 nm) for 20 min. The Rh complex was then removed occur predominately in a sequence of the genome termed - by washing with PBS, and 50000 cells were plated and the control region or d-loop (20). Nucleotides 303 315 allowed to grow. The procedure was repeated 4 days later within the control region (sequence 3′-G AG ) are especially 7 5 when cells had reached ∼50% confluency. Following the prone to insertions and deletions as well as dG to dT second round of photolysis, cells were again allowed to grow mutations (21). Nucleotides 303-315 are termed conserved to ∼50% confluency, and total DNA was purified using the sequence block II, a regulatory element that is involved in DNeasy kit (Qiagen) and eluted from the column in 2.5 mM primer formation for mitochondrial DNA replication (22, 23). Tris (pH 8.5). The DNA was amplified by the addition of Importantly, base oxidation arising from DNA charge × × transport has been shown both in vitro and in HeLa cells to 20 µLof2 reaction mix (2 Taq buffer, 4 mM MgCl2, damage predominately this conserved sequence, block 400 µM dNTP, 1 µM primer, and 10 units of Taq) to 20 µL ° II (24, 25). We hypothesize that, through DNA charge of each DNA sample.
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