Excision of nucleoside analogs in mitochondria by p53 protein

Abstract

Objective: 

Nucleoside analogs, used against HIV, can be incorporated into a mitochondrial DNA by DNA polymerase γ. Both the decrease in mitochondrial DNA and increased mutations of mitochondrial DNA may lead to mitochondrial diseases. The tumor suppressor protein p53 exhibits 3′→5′ exonuclease activity and can provide a proofreading function for DNA polymerases. In the present study, we investigated the ability of p53 to excise incorporated nucleoside analogs from DNA in mitochondria.

Design: 

The functional interaction of p53 and DNA polymerase γ during the incorporation of nucleoside analog was examined in mitochondrial fractions of p53-null H1299 cells, as the source of DNA polymerase γ.

Methods: 

Primer extension reactions were carried out to elucidate the incorporation and removal of nucleoside analogs.

Results: 

The results demonstrate that the excision of incorporated nucleoside analogs in mitochondrial fractions of H1299 cells increased in the presence of purified recombinant p53, or cytoplasmic extracts of large cell carcinoma 2 cells expressing endogenous wild-type p53 (but not specifically predepleted extracts) or cytoplasmic extracts of H1299 cells overexpressing wild-type p53, but not exonuclease-deficient mutant p53-R175H. The amount of nucleoside analogs incorporated into the elongated DNA with mitochondrial fractions of human colon carcinoma 116 (HCT116)(p53+/+) cells was lower than that of HCT116(p53−/−) cells. Furthermore, mitochondrion-localized elevation of p53 in HCT116(p53+/+) cells, following the irradiation-stress stimuli, correlates with the reduction in incorporation of nucleoside analogs and wrong nucleotides.

Conclusion: 

p53 in mitochondria may functionally interact with DNA polymerase γ, thus providing a proofreading function during mitochondrial DNA replication for excision of nucleoside analogs and polymerization errors.

Introduction

Mitochondrial toxicity from nucleoside reverse transcriptase inhibitors (NRTIs) therapy occurs in as much as 15–20% of the treated population ^[1]. NRTIs, serving as alternative substrates for HIV-1 reverse transcriptase, result in chain termination of viral DNA synthesis. The same nucleoside analogs, in addition to the target viral polymerase in cytoplasm, can be incorporated into a mitochondrial DNA (mtDNA) by DNA polymerase γ (pol γ), leading to termination of mtDNA synthesis and subsequent mitochondrial dysfunction ^[2]. NRTI therapy may be associated with increased risk of mtDNA mutation, the pathogenesis of which is likely to be multifactorial, involving increased oxidative stress and defective repair ^[3,4]. NRTI therapy-based mitochondrial toxicity is cumulative, increasing with duration of exposure in various tissues ^[5]. Hence, it is an important clinical problem with long-term significance to AIDS patients.

Acquired mitochondrial toxicity occurs as a consequence of incorporation of nucleoside analog into mtDNA or inhibition of mtDNA replication or both ^[1]. DNA pol γ is the sole polymerase in the mitochondria of eukaryotic cells, responsible for both DNA synthesis and repair ^[6]. DNA pol γ, encoded by the nuclear genome, contains two subunits, a large 125–140 kDa subunit, containing catalytic activities for polymerase and exonuclease, and a smaller accessory subunit of 41–55 kDa required for processive synthesis. The polymerase activity is essential for mtDNA maintenance, and its exonuclease activity is essential for mtDNA integrity ^[7–9]. DNA pol γ is unique among the cellular DNA polymerases in that it is highly sensitive to inhibition by NRTIs ^[1]. The high toxicity of dideoxy compounds may be caused by high rates of incorporation of the nucleoside analogs into mtDNA and the persistence of these analogs in mtDNA due to inefficient excision ^[10,11]. All of the currently approved antiviral nucleoside analogs were incorporated into DNA by pol γ, and all inhibited DNA synthesis in vitro to varying degrees. DNA pol γ was able to incorporate the analogs in the following order of efficiency: dideoxy nucleoside triphosphate (ddNTP) >3′-deoxy 2′,3′-didehydrothymidine-5′-triphosphate (D4T-TP) >carbovir-triphosphate (CBV-TP) >lamivudine triphosphate (3TC-TP) >azidothymidine triphosphate (AZT-TP) ^[12]. Interestingly, 3TC-TP is one of the analogs least likely to be incorporated and one of those most efficiently removed, which may explain the low mitochondrial toxicity induced by 3TC in vivo. Conversely, AZT-TP is the least likely to be incorporated into DNA by pol γ, but not efficiently removed from DNA. It was suggested that the failure of 3′→5′ exonuclease activity of pol γ to efficiently remove chain terminators might play a role in mitochondrial toxicity of nucleoside analogs ^[10–12].

The removal of the incorporated nucleoside analog from the 3′-end of DNA by 3′→5′ exonuclease-proficient DNA polymerase or by external proofreading activity associated with some polymerases or proteins may be viewed as a potential cellular mechanism of resistance to anticancer and antiviral drugs ^[13,14]. DNA synthesis/repair, proceeding in nucleus-free mitochondria, relies upon a preassembled DNA replication machinery of pol γ and multiple proteins to maintain mtDNA integrity. Therefore, it is of interest to identify the cellular components in mitochondria responsible for the removal of the mispairs and drugs from DNA.

The tumor suppressor protein p53 plays a crucial role in the cellular responses to various stress stimuli ^[15]. p53 induces either growth arrest (which prevents the replication of damaged DNA), or apoptosis (which is important for eliminating defective cells), or DNA repair. p53 elicits various biochemical activities that are directly related to its function in preserving the integrity of the cell's genetic information ^[16]. It displays intrinsic 3′→5′ exonuclease activity and by excision of the wrong incorporated nucleotides may provide a proofreading function for various DNA polymerases ^[17–22]. Furthermore, p53 might be involved in the repair of DNA damage caused by the incorporated nucleoside analogs ^[23,24].

Mitochondrial localization of p53 has been observed in stressed and unstressed cells ^[25,26]. Several studies ^[27] showed that the p53 is localized in mitochondria, associated with an inner membrane, the subfraction in which mtDNA is located. It exerts physical and functional interactions with mtDNA and pol γ and enhances base excision repair through direct interaction with the repair complex ^[28–30]. Our recent studies ^[31] showed that p53 in mitochondria, provided by recombinant or endogenous p53, increases the excision of the wrong nucleotides incorporated into DNA. The observations that p53 in mitochondria interacts with mtDNA and pol γ, taken together with the ability of p53 to remove incorporated nucleoside analog, led us to elucidate the capacity of p53 to catalyze the excision of nucleoside analog from the 3′-end of DNA in mitochondria. Our results demonstrate that a terminally incorporated nucleoside analog may be removed by p53 in mitochondria.

Materials and methods

Materials

The recombinant purified glutathione-S-transferase (GST) and p53-GST fusion proteins expressed in Escherichia coli were obtained from LabVision Corporation (Thermo Fisher Scientific, Fremont, California, USA).

Cell lines and culture medium

H1299 cells were grown in the presence of Rosewell park memorial institute medium (RPMI) 1640 and 10% fetal calf serum (FCS). The large cell carcinoma 2 (LCC2) cells (subclone derived from MCF-7 human breast cancer cells) were grown in Dulbecco's modified Eagle's medium (DMEM) lacking phenol red and 10% charcoal stripped FCS (CSS). The colorectal cancer cells isogenic for p53, human colon carcinoma 116 (HCT116)(p53+/+) and HCT116(p53−/−), were grown in McCoy's medium supplemented with 10% FCS. All lines were maintained in an atmosphere of humidified 95% air and 5% CO₂.

Template-primers

The sequences of the template-primers used for the experiments are depicted in figures. The primers were labeled at the 5′-end with T4 polynucleotide kinase (Fermentas AB, Vilnius, Lithuania) and [γ-³²P] ATP and annealed to the template DNA as described ^[19].

Preparation of mitochondrial fractions

Mitochondria were prepared as described ^[25]. Briefly, cells washed in TD buffer (135 mmol/l NaCl, 5 mmol/l KCl, 25 mmol/l Tris-HCl, pH 7.6) were centrifuged [2500 revolutions/min (rpm) at 4°C], resuspended in MgRSB buffer (1.5 mmol/l MgCl₂, 10 mmol/l NaCl, 10 mmol/l Tris, pH 7.5) and incubated for 10 min. Swollen cells were disrupted in a glass Dounce homogenizer. Mitochondria were pelleted from the washed homogenate, resuspended in a manitol–sucrose buffer, layered over a 1–2 mol/l sucrose step gradient and spun at 4°C for 30 min at 22 000 rpm. The mitochondria were collected from the 1.0–1.5 mol/l sucrose interphase.

Western blotting

Equal amounts of total protein of mitochondrial or cytoplasmic fractions were subjected to western blotting analysis ^[32]. The following antibodies were used: Do-1 monoclonal antibody for p53 (Oncogene Research Products, San Diego, California, USA), monoclonal antibody for cytochrome-c (Oncogene Research Products) and polyclonal antibody for c-jun (Santa Cruz Biotechnology, Santa Cruz, California, USA). Immunoprecipitation for p53 was performed as described ^[32].

Exonuclease/polymerase-coupled assays

DNA primer extension assay allowed simultaneous detection of both degradation (excision) and extension (polymerization). The incubation mixture (10 μl) contained 50 mmol/l Tris HCl (pH 7.5), 5 mmol/l MgCl₂, 1 mmol/l dithiothreitol (DTT), 0.1 mg/ml BSA, 5′-end labeled substrates, nucleoside analog and mitochondrial or cytoplasmic protein extracts. The variables, including reaction time and amounts of extracts, are given in the legends to figures. The reaction products (polymerization or excision) were analyzed by electrophoresis through 16% PAGE and detected by autoradiography ^[20].

Proteinase K treatment

Mitochondrial extracts were treated with 50 ng/ml proteinase K with or without 0.5% Triton X-100 (JT Baker Inc., Phillipsburg, New Jersey, USA) at room temperature for 30 min. The reaction was stopped by the addition of 0.1 mmol/l protease inhibitors mixture and then analyzed by western blotting with antibodies to p53 or B cell leukemia/lymphoma 2 (Bcl-2).

Results

The excision of nucleoside analog from DNA was elucidated using mitochondrial fractions of H1299 (p53-null) cells (H1299mit), as a model system. The composition and purity of the mitochondrial fractions were confirmed by the absence and presence of nuclear and mitochondrial markers, for example, c-jun and cytochrome-c, respectively (Fig. 1a). The lack of the mitochondrial locations of endogenous p53 was verified by western blotting analysis with specific antibodies against p53.

Fig. 1: The incorporation and excision of nucleoside analog in mitochondrial fractions of H1299 cells in the presence of p53. (a) Analysis of p53 levels in various fractions of H1299 and LCC2 cells by western blotting. Protein samples (20 μg) from mitochondrial (mit), nuclear (nuc) and cytoplasmic (cyt) fractions of the cells were subjected to SDS-PAGE. p53 protein expression was detected by the Do-1 antihuman p53 mAb. The distribution of the nuclear marker c-jun or the mitochondrial marker cyt-C was analyzed to ascertain the purity of each fraction. (b) Experimental scheme for analysis of nucleoside analog excision in mitochondrial extracts. The correctly paired 5′-end labeled template-primer was incubated with ddNTP and H1299mit for 60 min. After initial 60 min incubation, aliquots were taken for analysis of incorporation of nucleoside analog by PAGE, and the reaction mixture was further incubated in the absence or presence of p53. Aliquots were taken at various times and analyzed by PAGE. (c) The excision of the incorporated ddATP opposite the template T was examined with H1299mit and correctly paired template-primer (lane 1). After incorporation of ddATP (lane 2), the reaction mixture was further incubated in the absence (lane 3) or presence of p53-GST fusion protein (250 ng) (lanes 4–7) or GST protein (400 ng) (lanes 8 and 9). Aliquots taken after 20 min (lanes 4, 6 and 8) or 40 min incubation (lanes 3, 5, 7 and 9) were tested on 16% polyacrylamide gel. The positions of the 16mer and 17mer primers are indicated by arrows. (d) The excision of the incorporated ddATP opposite the template T [sequence of the template-primer (see Fig.1c)] with H1299mit (lane 1) was examined by further incubation of the reaction mixture in the absence or presence of p53-GST (250 ng) and of 10-fold molar excess of unlabeled homologous dsDNA. Lane 2 – incubation without p53-GST. Lane 3 – incubation without p53-GST in the presence of unlabeled dsDNA. Lane 4 – incubation with p53-GST. Lane 5 – incubation with p53-GST and unlabeled dsDNA. Aliquots were taken after 40 min incubation. The position of the 16mer primer is indicated by an arrow. ddNTP, dideoxy nucleoside triphosphate; dsDNA, double-stranded DNA; GST, glutathione-S-transferase; H1299mit, H1299 mitochondria; LCC2, large cell carcinoma 2.

The excision of the incorporated nucleoside analog in mitochondrial extracts in the presence of purified recombinant p53

The impact of the p53 on the incorporation and excision of the nucleoside analog during DNA synthesis by pol γ in mitochondria was tested in a sequential reaction experiment (Fig. 1b). The strategy was to follow after the excision of the incorporated nucleoside analog in the absence or presence of p53. The results show that pol γ in mitochondria, using standing-start template-primer, was able to use ddATP as a substrate for incorporation, thus creating a 17mer substrate (representing incorporation of nucleoside analog) for the excision step (Fig. 1c, lane 2). After initial 60 min incubation, the reaction mixture was further incubated in the absence or presence of recombinant p53-GST fusion protein. The accumulation of 17mer product in the absence of p53-GST indicates that ddATP was inefficiently corrected by exonuclease-proficient mitochondrial pol γ (lane 3). However, the addition of the recombinant p53-GST increased the excision of incorporated nucleoside analogs. The intensity of the 17mer band decreased with the amount of p53 protein added to the assays and with incubation time (lanes 4–7). In the control experiment, there was no reduction of 17mer product in the presence of GST protein alone, indicating that the GST moiety did not make a significant contribution to the observed increased excision of nucleoside analog (lanes 8 and 9). These results were reproduced three times with separate preparations of mitochondrial extracts.

Excision of the incorporated nucleoside analog may occur by either an intrinsic intramolecular mechanism (without dissociation of the DNA from the enzyme) or an external intermolecular mechanism (following the release of the DNA from the enzyme) ^[33,34]. The incorporated nucleoside analog leads to termination of the growing DNA strand and probably to the dissociation of the enzyme from the template-primer. To elucidate the involvement of external p53 in the excision of the incorporated nucleoside analog and to test whether the nucleoside analog–DNA might dissociate from the enzyme before excision, the reaction mixture was further incubated without or with p53-GST in the absence or presence of unlabeled competitor double-stranded DNA (dsDNA). The low efficiency of ddATP excision by pol γ alone (Fig. 1d, lane 2) was unaffected by the presence of unlabeled homologous DNA (lane 3). However, although incubation with p53-GST resulted in efficient excision of the incorporated ddATP and decrease of 17mer product (lane 4), the substantial reduction in exonucleolytic excision of the ddATP was observed in the presence of unlabeled dsDNA (lane 5). Thus, our experimental data suggest that following the incorporation of the nucleoside analog, the 17mer product dissociates and may be accessible for excision by external p53 exonuclease.

The excision of the incorporated nucleoside analog in mitochondrial extracts in the presence of endogenous p53

Sequential incorporation and excision of various nucleoside analogs by pol γ in H1299mit was further assessed with two DNA/DNA substrates in the presence of cytoplasmic extract of LCC2 cells (LCC2cyt), expressing endogenous wild-type p53 (wtp53) (Fig. 2a, lane 1) with intrinsic exonuclease activity ^[32]. The accumulation of 17mer product following the incorporation of ddATP opposite the template T (I) (Fig. 2b, lane 1) or dideoxy thymidine triphosphate (ddTTP) opposite the template A (II) (Fig. 2b, lane 4) was observed with standing-start substrates. The incorporation of cytosine arabinoside (Ara-C) opposite the G and production of 19mer product was detected in a running-start reaction in the presence of dATP and Ara-C (III) (Fig. 2b, lane 7), following the incorporation of two running-start A's opposite two T residues in the template. The addition of the LCC2cyt to all these reactions substantially reduced the number of incorporated nucleoside analogs (either ddATP, ddTTP or Ara-C) (Fig. 2b, lanes 3, 6 and 9, respectively). Indeed, the decrease in the amount of 17mer products (with standing-start template-primer) and of 19mer products (with running-start template primer) was observed concomitant with the appearance of lower products (15mer, 14mer and more). In control experiments, we verified no excision of incorporated nucleoside analog with p53-null H1299cyt (lanes 10–12).

Fig. 2: The incorporation and excision of various nucleotide analogs in H1299 mitochondrial fractions in the presence of cytoplasmic fraction of LCC2 cells. (a) p53 expression was tested in LCC2cyt (10 μg) before (lane 1) and after immunoprecipitation by the Do-1 antihuman p53 mAb (lane 2) or antihorse IgG (H + L) (lane 3) by WB. Actin was blotted as a protein loading control. (b) The incorporation of various NAs was examined with H1299mit (5 μg): ddATP opposite the template T (template-primer I) (lanes 1–3), ddTTP opposite the template A (template-primer II) (lanes 4–6) or Ara C opposite the template G (template-primer III) (lanes 7–9). After the initial incorporation of the NAs (60 min incubation) (lanes 1, 4 and 7), the reaction mixtures were further incubated in the absence (lanes 2, 5 and 8) or presence of 5 μg LCC2cyt (lanes 3, 6 and 9) or H1299cyt (lanes 10–12). Aliquots were taken after 40 min incubation. (c) After the incorporation of ddATP (template-primer I, Fig.2b) (lane1), the reaction mixture was further incubated in the absence (lane 2) or presence of 5 μg of LCC2cyt (lane 3) or LCC2cyt immunodepleted by antihorse IgG (H + L) (lane 4) or by the Do-1 antihuman p53 mAb (lane 5). Aliquots were taken after 40 min incubation. (c) Excision of the incorporated ddATP in H1299mit (lane 1) was examined in the presence of H1299cyt (1 μg) transfected with control vector (lane 2), mutant p53-R175H expression vector (lane 3) or wtp53 (lanes 4 and 5). Aliquots were taken after 20 min (lanes 2–4) and 40 min (lane 5) incubation. The positions of the 16mer and 18mer primers are indicated by arrows. Ara-C, cytosine arabinoside; ddTTP, dideoxy thymidine triphosphate; H1299cyt, cytoplasmic fractions of H1299 cells; H1299mit, H1299 mitochondria; IgG, immunoglobulin G; LCC2cyt, cytoplasmic large cell carcinoma 2; NAs, nucleotide analogs; WB, western blotting.

To confirm that the nucleoside analog excision activity detected with LCC2cyt was attributed to p53, we immunodepleted the p53 from the LCC2cyt in order to analyze whether it would abolish the exonuclease function of the protein. The results obtained show that the depletion of p53 protein from LCC2cyt by Do-1 antip53 antibody (Fig. 2a, lane 2) led to a significant decrease in nucleoside analog excision activity (Fig. 2c, lane 5). Conversely, nonspecific immunoprecipitation by antihorse immunoglobulin G (IgG) (H + L) (Fig. 2a, lane 3) did not affect exonuclease activity (Fig. 2c, lane 4). Thus, the inhibition of excision reaction was probably due to immunoprecipitation of p53 protein.

Our studies ^[35] illustrated that cytoplasmic fractions of H1299 cells overexpressing wtp53, but not exonuclease-deficient mutant p53-R175H, had higher levels of exonuclease activity than those transfected with the control vector. Furthermore, increased removal of incorporated wrong nucleotide was observed in mitochondrial fraction in the presence of H1299cyt overexpressing wtp53, but not exonuclease-deficient mutant p53-R175H ^[31]. These cytoplasmic fractions were used to further define the contribution of endogenous p53 exonuclease to the excision of incorporated nucleoside analog in H1299mit. The results showed the efficient excision of the incorporated ddATP in H1299mit in the presence of H1299cyt overexpressing wtp53 (Fig. 2d, lanes 4 and 5). No efficient excision was observed in the presence of H1299cyt transfected with the control vector (lane 2) or exonuclease-deficient mutant p53-R175H (lane 3).

In-vivo correlation between mitochondrial p53 and enhanced exonuclease activity

Mitochondrial localization of p53 has been observed in several systems. To elucidate the impact of the endogenous p53 in mitochondria, the incorporation and excision of nucleoside analog in DNA was examined in mitochondrial extracts derived from isogenic HCT116 cells. p53 was detected in mitochondrial fraction of HCT116(p53+/+) cells, but not of HCT116(p53−/−) cells by western blotting analysis with Do-1 antihuman p53 mAb (Fig. 3a).

Fig. 3: Incorporation of nucleoside analog in mitochondrial fractions of HCT116 cells. (a) Mitochondrial fractions (10 μg) of HCT116(p53−/−) or HCT116(p53+/+) cells were examined for p53 expression by Do-1 anti-p53 mAb. Cytochrome-c was blotted as a protein loading control. (b) Mitochondrial extracts of p53(−/−) (lanes 1 and 2) and p53(+/+) cells (lanes 3 and 4) were tested for 3′→5′ exonuclease activity with dsDNA substrates containing 3′-terminal mispair A: A. Aliquots were taken after 20 min (lanes 1 and 3) and 40 min (lanes 2 and 4) incubation. (c) Mitochondrial extracts of p53(−/−) and p53(+/+) cells were tested for incorporation of either ddATP (lanes 1 and 2), or wrong nucleotide dGTP (lanes 3 and 4), or correct dATP (lanes 5 and 6) opposite the template T. The position of the 16mer primer is indicated by an arrow. Cyt c, cytochrome c; dGTP, deoxyguanosine triphosphate; dsDNA, double-stranded DNA; HCT, human colon carcinoma.

Higher A: A mispair excision activity was observed in p53(+/+)mit (Fig. 3b, lanes 3 and 4) relative to that detected in equivalent amounts of p53(−/−)mit (lanes 1 and 2). Remarkably, both fractions had comparable DNA synthesis activity during the incorporation of correct dATP nucleotides (Fig. 3c, lanes 5 and 6). However, the incorporation of ddATP or wrong nucleotide-deoxyguanosine triphosphate opposite the template T with p53(+/+)mit was lower than with p53(−/−)mit (lanes 1–4). Thus, the reduction in incorporation of nucleoside analog or wrong nucleotide with p53(+/+)mit is not due to the decrease in polymerization activity of pol γ in p53(+/+)mit, but rather due to the presence of p53.

p53 may exert the functional heterogeneity in its noninduced and activated states. To further substantiate the importance of p53 in mitochondria, we assessed p53 exonuclease activity in the context of a cellular stress. Irradiation induced the mitochondrial translocation of p53 in various cell lines (e.g. MCF-7 and HCT116) that are resistant to apoptosis ^[36]. The absence of activated apoptotic pathway in irradiation-HCT116(p53+/+) cells (i.e. under p53 inducible conditions) permits the use of these mitochondrial fractions as a suitable experimental system. Irradiation-induced p53 translocation to the mitochondria was demonstrated by western blotting analysis (Fig. 4a). The localization of p53 within mitochondrial extracts was evaluated by proteinase K protection assay. Western blotting analysis of irradiation-(p53+/+)mit treated with proteinase K in the absence or presence of Triton X-100 showed that Bcl-2, which is associated with the external mitochondrial membrane, was completely degraded, regardless of whether Triton X-100 was added or not (Fig. 4b). However, p53 protein was protected from proteinase K in the absence of detergent, indicating that it was located in the inner mitochondrial compartment.

Fig. 4: The incorporation of nucleoside analog decreases in mitochondrial fraction of irradiation-treated HCT116(p53+/+) cells. HCT116(p53+/+) cells were exposed to ionizing radiation (20 gray) using a Gammacell 1000 Elite. (a) WB analysis of mitochondrial fractions (10 μg) of untreated and IR-treated cells for the status of p53 (24 h post-IR). The mitochondrial protein cyt-c was used as a loading control. (b) p53 detection in submitochondrial fraction. IR-treated p53(+/+)mit (control-C) was treated with 50 ng/ml PK in the absence or presence of 0.5% TX at room temperature for 30 min. Reactions were tested by WB with antibodies antip53 or anti-Bcl-2. (c) Mitochondrial extracts of IR-untreated and treated (p53+/+) cells (24 h post-IR) were examined for 3′-terminal A: A mispair excision activity (exo) (lanes 1 and 2) (template-primer-set I) and for incorporation of either ddCTP-nucleoside analog (inc NA) (lanes 3–4) or correct dATP (polym) (lanes 7 and 8) (template-primer set II). Mitochondrial extracts of IR-untreated or treated (p53−/−) cells (24 h post-IR) were examined for incorporation of correct dATP (lanes 5 and 6). The positions of the 16mer and 18mer primers are indicated by arrows. Bcl-2, B cell leukemia/lymphoma 2; cyt c, cytochrome c; ddCTP, dideoxy cytosine triphosphate; exo, exonuclease; HCT, human colon carcinoma; inc NA, incorporated nucleoside analog; IR, irradiation; PK, proteinase K; polym, polymerase; TX, triton; WB, western blotting.

The analysis of the mitochondrial fractions for the exonuclease activity revealed the increase in constitutive A: A mispair excision capacity following the irradiation treatment and p53 translocation to mitochondria (Fig. 4c, lanes 1 and 2). Increased level of p53 in irradiated-(p53+/+)mit was associated with decreased incorporation of dideoxy cytosine triphosphate (ddCTP) opposite the template G in a running-start reaction in the presence of dATP and ddCTP following the incorporation of two running-start A's (lanes 3–4). Apparently, pol γ was not responsible for the observed reduction in incorporation of nucleoside analog in p53(+/+)mit following the irradiation event, as there was no difference in correct polymerization activity between mitochondrial fractions of irradiation-untreated and treated HCT116(p53−/−) and HCT116(p53+/+) cells (lanes 5–8).

Discussion

The overall toxicity of a nucleoside analog will be a function of the frequency of incorporation of the nucleoside analog and excision by the proofreading exonuclease ^[1]. Following the incorporation of nucleoside analog, the dissociation of polymerase from DNA may allow nucleoside analog–DNA to bind external exonuclease for removal of nucleoside analog. The purpose of the present study was to test the ability of p53 protein in mitochondria to serve as an external proofreader for pol γ. Several lines of experimental evidence demonstrate the increased excision of the incorporated nucleoside analogs in H1299mit in the presence of p53: First, excision of incorporated nucleoside analog increases in the presence of recombinant purified p53. Second, excision of incorporated nucleoside analog increases in the presence of p53 provided by LCC2cyt expressing endogenous wtp53. The enhanced exonuclease activity was greatly reduced in predepleted LCC2cyt with specific anti-p53 antibodies. Third, increased removal of nucleoside analog was observed in H1299mit in the presence of H1299cyt overexpressing wtp53, but not exonuclease-deficient mutant p53-R175H. Fourth, the incorporation of nucleoside analog is lower in p53(+/+)mit compared with that in p53(−/−)mit, although p53 status did not influence the incorporation of correct nucleotide. Fifth, mitochondrion-localized elevation of p53 following the irradiation-stress stimuli correlates with the increased mispair excision activity and low incorporation of nucleoside analog. The fact that p53 localizes to the mitochondria and interacts with mtDNA and pol γ, taken together with our observations that the presence of p53 (provided by recombinant or endogenous p53) reduces the amount of incorporation of nucleoside analog in H1299mit, suggests that p53 may potentially participate in nucleoside analog excision.

Mitochondrial toxicity may be caused by termination of the growing nascent DNA strand after incorporation of the nucleoside analog into mtDNA or by inhibition of pol γ exonucleolytic proofreading ^[1]. Increased mtDNA mutations were demonstrated in patients undergoing NRTI therapy, in which novel mtDNA sequence variations occurred over a relatively brief period (∼24 months) ^[4]. Inhibition of pol γ proofreading by monophosphates results in as much as a 20-fold decrease in replication fidelity in vitro^[6]. Interestingly, azidothymidine-monophosphate (AZT-MP) is known to accumulate in millimolar concentrations in cells, whereas the concentrations of the triphosphate form are found to be only 2 micromolar ^[1]. AZT-MP, by binding to the exonuclease active site of pol γ, results in lower fidelity and excessive accumulation of mutations within mtDNA. The fact that p53 may improve the accuracy of DNA synthesis by the excision of mispaired nucleotides (thus preventing the production of polymerization errors), in addition to excision of nucleoside analog (thus decreasing their potential for chain termination), implies that this cellular error-correction pathway may compensate for a lack of effective proofreading for pol γ-induced replication errors (Fig. 5).

Fig. 5: Error corrections in mitochondria in the presence of p53. In mitochondria, NA may be incorporated into mtDNA, thus resulting in chain termination, or binds to the exonuclease active site of pol γ, thus leading to inhibition of exonuclease function and increase in mutations. The excision of the NA by p53 protein may decrease their potential for chain termination, and the excision of the wrong nucleotide may increase the fidelity of DNA synthesis by pol γ. mtDNA, mitochondrial DNA; NA, nucleoside analog.

p53 is able to elicit a spectrum of different biological effective pathways depending on the subcellular localization of the protein. The proofreading efficiency and excision of nucleoside analog may increase when decreasing the ratio between polymerase/exonuclease ^[37]. The antimutagenic capacity will be enhanced through increase in total p53 concentration or p53 targeting or both. Mitochondrial p53 levels are proportional to total p53 levels, and a small fraction of protein associated with mtDNA in the absence of exogenous stress is independent of apoptosis ^[38]. Both HIV infection and NRTIs may contribute to mitochondrial oxidative stress and dysfunction. In HIV-infected cells, probably two stress conditions, reverse transcription process in cytoplasm and integration of proviral DNA within cellular DNA in nucleus ^[39], may trigger distinct signaling pathways in controlling p53 subcellular localization. In primary cells, p53 levels were increased by 24 h after HIV-1 infection ^[40]. Hence, it is reasonable to speculate that in HIV-infected cells there may be an increase in local mitochondrial concentration of p53. p53 in mitochondria probably has a transient interaction with replication complex; the DNA synthesis may be a dynamic process with p53-component binding and dissociating the polymerization complex during DNA synthesis, thus affecting the polymerase (pol γ)/exonuclease (p53) ratio. Consequently, the decrease in the ratio of pol γ/p53 due to the increase in local p53 concentration in mitochondria may enhance the proofreading efficiency and excision of nucleoside analog by external p53.

Interestingly, HIV induces lymphocyte apoptosis by a p53-initiated mitochondrial-mediated mechanism ^[40]. It is tempting to hypothesize that p53 in mitochondria may serve a dual purpose; in addition to the p53-dependent apoptosis, the protein may be involved in excision of wrong nucleotide or nucleoside analog. Future studies should evaluate the location of p53, potential participation in mtDNA repair and interaction of p53 with other proteins in various compartments of the virus-infected cells.

p53 is a multifunctional protein with positive and negative effects. In general, drug resistance that occurs in cancer chemotherapy and antiviral therapy is a negative event that will decrease the efficacy of the treatment. The recognition and removal of nucleoside analog from cellular and viral DNAs by p53 exonuclease activity in various compartments of the cell may play a role in decreasing drug activity, leading to various biological outcomes: the removal of the incorporated difluorodeoxycytidine monophosphate (dFdCMP) from DNA was detected in whole cells with wtp53 (ML-1), but not in CEM cells harboring mutant p53 ^[23]. The excision of the incorporated nucleoside analog from DNA in nucleus may confer resistance to the drugs (negative effect); the removal of the nucleoside analog by p53 from DNA incorporated by HIV-1 reverse transcriptase in cytoplasm may confer resistance to the drugs (negative effect) ^[24]; and the excision of nucleoside analogs from mitochondrial DNA may decrease the potential for chain termination and host toxicity (positive effect). Apparently, the presence of p53 in mitochondria may be important, as the excision of the mispair and nucleoside analog by p53 is favorite event for mitochondrial function.

Currently available NRTIs differ in their convenience of administration, resistance profile and frequency and severity of adverse effects. Clinical toxicities due to the mitochondrial dysfunction induced by NRTIs (e.g. lactic acidosis, pancreatitis and neuropathy) may limit certain treatment regimens ^[5]. NRTIs therapy may lead to both generalized and tissue-specific toxicities. One of the proposed mechanisms by which NRTIs interfere with mitochondrial functions is the depletion of mtDNA due to the inhibition of pol γ. NRTIs inhibit both HIV-1 reverse transcriptase in cytoplasm (antiviral activity) and pol γ in mitochondria (host toxicity). NRTI import, compartmentalization and phosphorylation to active moieties by cellular kinases are key factors for inhibition of pol γ. Knowledge of the mechanism of inhibition of pol γ may be utilized to obtain selectivity for HIV-1 reverse transcriptase over pol γ. The possibility that p53 exonuclease may remove the incorporated NRTI suggests that the presence of the cellular component – p53 in mitochondria may be important in defining the cytotoxicity of NRTIs toward mtDNA replication, thus affecting risk–benefit approach (NRTI toxicity versus viral inhibition).

Acknowledgements

This research was supported by a grant from the Israel Cancer Association. The authors thank Novitsky Elena for excellent technical assistance.

Author's contribution: S.H. and E.B. performed the experiments; G.R. critically edited the manuscript; M.B. designed the project and wrote the manuscript.