Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication*

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol γ), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol γ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g→t and T885S, c.2653a→t). Biochemical characterization of purified, recombinant forms of pol γ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50–70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol γ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol γ and examines the consequences of mitochondrial disease mutations in this region.

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol ␥), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol ␥ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g3a; T851A, c.2551a3g; R852C, c.2554c3t; R853Q, c.2558g3a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g3t and T885S, c.2653a3t). Biochemical characterization of purified, recombinant forms of pol ␥ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50 -70% wildtype polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol ␥ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol ␥ and examines the consequences of mitochondrial disease mutations in this region.
As the only DNA polymerase found in animal cell mitochondria, DNA polymerase ␥ (pol ␥) 3 bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1,2). Mammalian cell pol ␥ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3Ј 3 5Ј exonuclease, and 5Ј-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol ␥ complex to DNA (4,5).
Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol ␥, is frequently involved in disorders linked to mutagenesis of mtDNA (8,9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and recessive forms of progressive external ophthalmoplegia (PEO), Alpers syndrome, parkinsonism, ataxia-neuropathy syndromes, and male infertility (tools.niehs.nih.gov/polg) (9).
Alpers syndrome, a hepatocerebral mtDNA depletion disorder, and myocerebrohepatopathy are rare heritable autosomal recessive diseases primarily affecting young children (10 -12). These diseases generally manifest during the first few weeks to years of life, and symptoms gradually develop in a stepwise manner eventually leading to death. Alpers syndrome is characterized by refractory seizures, psychomotor regression, and hepatic failure (11,12). Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9,14,15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16,17).
During the initial cloning and sequencing of the human, Drosophila, and chicken pol ␥ genes, we noted a highly conserved region N-terminal to motif A in the polymerase domain that was specific to pol ␥ (18). This region corresponds to part of the thumb subdomain that tracks DNA into the active site of both Escherichia coli pol I and T7 DNA polymerase (19 -21). A high concentration of disease mutations, many associated with Alpers syndrome, is found in the thumb subdomain.
Here we investigated six mitochondrial disease mutations clustered in the N-terminal portion of the polymerase domain of the enzyme (Fig. 1A). Four mutations (G848S, c.2542g3a; T851A, c.2551a3g; R852C, c.2554c3t; R853Q, c.2558g3a) reside in the thumb subdomain and two (Q879H, c.2637g3t and T885S, c.2653a3t) are located in the palm subdomain. These mutations are associated with Alpers, PEO, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), ataxia-neuropathy syndrome, Leigh syndrome, and myocerebrohepatopathy (Table 1). Purified, recombinant forms of pol ␥ bearing these amino acid substitutions were biochemically characterized in vitro to evaluate polymerase activity, DNA binding, steady state kinetics of nucleotide insertion, primer extension activity, misinsertion fidelity, and association with the accessory subunit. Kinetic analysis revealed both nucleotide binding and catalytic defects for the thumb domain mutants, and mutations affecting the most highly conserved residues exhibited the most severe defects in overall catalysis. Nevertheless, none of the mutations reduced the in vitro nucleotide selectivity, and both physical and functional interactions with the accessory subunit were unaffected for each enzyme. Interestingly, DNA binding was impaired in only two of the four thumb domain mutants, suggesting a specific orientation of DNA within the polymerase active site.

EXPERIMENTAL PROCEDURES
Construction of Substituted p140 Proteins-Mutations in the cDNA encoding the exonuclease-deficient (Exo Ϫ ) pol ␥ (POLG) were generated using the QuikChange site-directed mutagenesis kit (Stratagene) with the pQVSL11.4 baculoviral transfer vector encoding p140 Exo Ϫ without its mitochondrial targeting sequence (22) as template. This exonuclease deficient pol ␥ without an additional mutation is denoted wild-type (WT) in this study. The oligonucleotides containing the point mutations (underlined nucleotide) for introducing the G848S mutation in POLG are 5Ј-CAA GTG GTG ACT GCC AGC  ACC ATC ACT CGC CGG-3Ј and 5Ј-CCG GCG AGT GAT  GGT GCT GGC AGT CAC CAC TTG-3Ј, for the T851A mutation are 5Ј-CTG CCG GCA CCA TCG CTC GCC GGG CTG  TGG AG-3Ј and 5Ј-CTC CAC AGC CCG GCG AGC GAT  GGT GCC GGC AG-3Ј, for the R852C mutation are 5Ј-GCC  GGC ACC ATC ACT TGC CGG GCT GTG GAG CCC-3Ј and  5Ј-GGG CTC CAC AGC CCG GCA AGT GAT GGT GCC  GGC-3Ј, for the R853Q mutation are 5Ј-GGC ACC ATC ACT  CGC CAG GCT GTG GAG CCC AC-3Ј and 5Ј-GTG GGC  TCC ACA GCC TGG CGA GTG ATG GTG CC-3Ј, for the  Q879H mutation are 5Ј-GTT GAA AGC CAT GGT GCA TGC  CCC ACC TGG CTA C-3Ј and 5Ј-GTA GCC AGG TGG GGC  ATG CAC CAT GGC TTT CAA C-3Ј, and for the T885S mutation are 5Ј-GCC CCA CCT GGC TAC TCC CTT GTG GGT GCT GAT G-3Ј and 5Ј-CAT CAG CAC CCA CAA GGG AGT AGC CAG GTG GGG C-3Ј. All six mutations were confirmed by DNA sequencing of the pol ␥ insert.
Expression and Purification-The WT and mutant forms of the His 6 affinity-tagged recombinant catalytic subunit of human pol ␥ were produced in baculovirus-infected Sf9 cells, and the proteins were purified to homogeneity as described previously (23, 24). The His 6 affinity-tagged p55 accessory subunit was expressed in E. coli and purified to homogeneity as described previously (25). After purification the proteins were frozen in small aliquots in liquid nitrogen and stored at Ϫ80°C.
The primer extension analysis of WT and mutant forms of p140 proteins utilized a 5Ј-32 P-end-labeled 35-mer oligonucleotide (5Ј-CCA GTG CCA AGC TTG CAT GCC TGC AGG TCG ACT CT-3Ј) singly primed M13 ssDNA substrate as described (23) without the preincubation step. Briefly, the 10-l reaction contained 25 mM HEPES-KOH (pH 7.6), 5 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.05 mg/ml heat-treated BSA, 0 or 75 mM NaCl, 25 M dNTPs, 20 fmol of the labeled oligonucleotide, 50 fmol of purified WT or mutant p140 enzyme in the presence or absence of 100 fmol of the p55 accessory subunit, as indicated. After incubation at 37°C for 20 min, reactions were terminated, and products were analyzed using denaturing polyacrylamide gel electrophoresis as described (23). Gels were dried, exposed to a phosphor screen, and visualized with a Typhoon 9400 PhosphorImager (Molecular Dynamics).
Nucleotide Misincorporation Kinetics-The fidelity of nucleotide selection by pol ␥ was determined with a polyacrylamide gel-based, single nucleotide extension assay utilizing a 22-mer oligonucleotide (5Ј-ACC ATG ACC ATG TAC ATC AGA G-3Ј (primer)) annealed to a 40-mer oligonucleotide (3Ј-TGG TAC TGG TAC ATG TAG TCT CAG CCT GCA TAT AGT CAC T-5Ј (template)) (24,26). Briefly, reaction mixtures (10 l) contained 25 mM HEPES-KOH (pH 7.6), 2 mM 2-mercaptoethanol, 0.1 mM EDTA, 50 g/ml acetylated BSA, 5 mM MgCl 2 , 1 pmol of primer-template, 50 fmol of exonuclease-deficient p140 with 100 fmol of p55 and one of the four common deoxynucleoside triphosphates. As the next correct nucleotide, dTTP concentration was varied from 0 to 640 nM, whereas the three incorrect nucleotide concentrations were varied from 0 to 3 mM. After incubation at 37°C for 10 min, reactions were terminated by the addition (10 l) of 95% deionized formamide and 10 mM EDTA. Samples (2 l) were boiled for 5 min at 95°C and resolved by electrophoresis on 12% polyacrylamide gels containing 6 M urea. Gels were dried as before, and radioactive bands were detected with a Typhoon 9400 PhosphorImager (Molecular Dynamics) and quantified with NIH Image software. K m and V max values were determined by fitting the data to the steady state Michaelis-Menten model using KaleidaGraph (Version 4.0, Synergy).
Circular Dichroism Measurements and Analysis-Circular dichroism (CD) studies were performed with a Jasco 810 Spectropolarimeter equipped with a Peltier thermal controller (Jasco, Inc., MD) following the method described in Chan et al. (17) with p140 proteins at 20 g/ml.
Immunoprecipitation Assay-Rabbit polyclonal antibodies raised against recombinant human p55 accessory subunit (4) were immobilized on protein G-Sepharose beads (GE Healthcare), and the beads were then equilibrated in phosphate-buffered saline Nonidet P-40-BSA buffer consisting of 50 mM potassium phosphate (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, and 0.1 mg/ml BSA. Prepared protein G-Sepharose beads (20 l) were mixed as indicated with purified p55 accessory subunit (3 g) and p140 WT or mutant proteins (3 g) in a 1.5-ml polypropylene microcentrifuge tube, and the reactions were brought to a final volume of 400 l with phosphate-buffered saline Nonidet P-40-BSA buffer. Samples were then processed as previously described (16,25), permitting visualization of coimmunoprecipitated proteins by immunoblot analysis.

Alpers Syndrome pol ␥ Mutations Located in the Thumb Subdomain of the Polymerase Domain Are Highly Conserved-Six
POLG mutations associated with Alpers syndrome or myocerebrohepatopathy were chosen for analysis because of their location in the thumb and early palm subdomains of the polymerase active site. The G848S, T851A, R852C, R853Q, Q879H, and T885S mutations were identified in trans with other POLG mutations in mitochondrial disease patients with variable clinical presentations (Table 1). These six mutations occurred in a region of pol ␥ with high amino acid homology among several organisms (Fig. 1B). Four of these six residues, Gly-848, Thr-851, Arg-852, and Arg-853, reside in close proximity to one another at the N terminus of the polymerase region and are invariant from yeast to humans. However, the Gln-879 and Thr-885 residues are only moderately conserved through evolution in the pol ␥ amino acid sequences. To gain insight into the consequences of these mutations in the associated mitochondrial diseases, all six mutant proteins were constructed in a pol ␥ Exo Ϫ background (D198A/E200A) (23) to abolish 3Ј 3 5Ј exonuclease activity that can interfere with biochemical assays involving nucleic acids. The WT and the mutant proteins were purified to homogeneity after overproduction in baculovirusinfected insect cells as previously described (23, 24).
Alpers Mutant p140 Proteins Retained Their Secondary Structure but Exhibited Decreased Stability-The structural integrity of these six mutant proteins was confirmed before biochemical characterization in vitro. The effects of these point mutations on p140 secondary structure were assessed by CD spectroscopy. As shown in Fig. 2A, the spectrum of each mutant protein followed a similar pattern to that of the WT enzyme, suggesting that they are properly folded and retain secondary structure similar to the WT enzyme. Also, the melting temperature (T m ) and the change in enthalpy for protein folding (⌬H m ) were calculated for each mutant protein by monitoring the thermal denaturation of ␣-helices over a 30 -60°C temperature range. This analysis revealed that all mutant proteins had similar T m values ranging between 45 and 49°C (Fig. 2B). However, all mutant proteins except T885S had a lower ⌬H m value compared with WT enzyme (Fig. 2B), suggesting that these mutations might affect the overall stability and, therefore, the biochemical properties of these enzymes.

Mutation of Highly Conserved Residues Has Drastic Effects on
Enzyme Activity-To determine the effect of the six mutations on overall enzyme activity, polymerase assays were performed using poly(dA)-oligo(dT) 12-18 as substrate, as previously described (24). The Gly-848, Thr-851, Arg-852, and Arg-853 amino acid residues are highly conserved and clustered in the thumb domain (Fig.  1B), suggesting their significance in the overall function of the enzyme. Indeed, the specific activity of the WT enzyme was 210 units/ng, whereas the specific activities of the G848S-, T851A-, R852C-, and R853Q-substituted proteins were less than 1 unit/ng (Fig. 3A). In contrast, mutation of the moderately conserved residues only reduced polymerase activity of the Q879H and T885S proteins to 53 and 68% of WT p140 activity, respectively (110 and 140 units/ng, respectively) ( Fig. 3A).
Polymerase activity was also determined using activated calf thymus DNA as substrate, which revealed a 15-fold reduction in WT p140 activity compared with the poly(dA)-oligo(dT) 12-18 substrate (14 versus 208 units/ng) (Fig. 3B). The mutant p140 proteins showed a similar substrate preference, although the activities of the T851A and R852C proteins were not as fully reduced on the natural DNA substrate.

DNA Binding Affinity of Mutant Proteins Suggests a Possible Orientation of the Enzyme with Primer-Template during
Catalysis-The reduced enzyme activity of these mutant p140 proteins can be because of their impaired binding to DNA. Hence, the DNA binding affinity of the WT and mutant p140 enzymes was estimated by electrophoretic mobility shift assay. Radiolabeled 34/38-mer oligonucleotide primer-template was incubated with various concentrations of WT or mutant p140, and native polyacrylamide gel electrophoresis was used to separate protein-DNA complexes from free DNA. The apparent K d(DNA) values were subsequently calculated from the reciprocal plots of the fraction of DNA shifted at various enzyme concentrations. This analysis revealed that the WT p140 enzyme had strong affinity to DNA (K d(DNA) ϭ 31 nM, Fig. 4A). Although most mutant proteins retained DNA binding affinity at the level of WT protein, the G848S (K d(DNA) ϭ 152 nM) and R852C (K d(DNA) ϭ 114 nM) p140 proteins exhibited 5-and 4-fold reductions, respectively, in DNA binding affinity compared with the WT enzyme (Fig. 4A). Gly-848 and Arg-852 are four residues apart in the primary sequence. Because the thumb subdomain in family A DNA polymerases is predominantly ␣-helical (19 -21), these two residues could be located on the same face of an ␣-helix that interacts with the DNA substrate. Using a helical wheel drawing program we determined that these residues would likely reside on the same helical face (Fig.  4B). Hence, mutations of these critical residues had greater effects on DNA binding than the other Alpers mutant proteins presented in this study.

Alpers Mutant Proteins Physically and Functionally
Interact with the p55 Accessory Subunit-Previous studies have shown that interaction of the accessory subunit (p55) with the p140 catalytic subunit enhances the processivity and DNA binding properties of the catalytic subunit (4), and the most common POLG mutation, namely the A467T, has been demonstrated to impair this association (16). Hence, the physical association of p55 with WT and mutant p140 enzymes was assessed with coimmunoprecipitation experiments. The p55 protein was linked to protein G-Sepharose beads with polyclonal antibodies raised against recombinant p55 protein, and the ability of this immobilized p55 to capture WT and mutant forms of p140 was screened. This analysis revealed that WT and all six mutant p140 enzymes interacted physically with p55 (Fig. 5, lanes  4 -10). The interaction was clearly dependent on p55, as no appreciable amount of WT p140 was immunoprecipitated in the absence of p55 (Fig. 5, lane 2).
Next, to check whether this physical interaction of the p140 mutant proteins with p55 translates into functional interaction, an in vitro primer extension assay was performed using WT and mutant p140 enzymes in the presence or absence of p55, as previously described (4). The processivity of the catalytic subunit is stimulated as much as 50-fold upon interaction with the accessory subunit (4). The assay revealed that WT p140 extended about 100 nucleotides in the absence of NaCl under conditions that permitted multiple binding events, and the activity was mildly inhibited in the presence of 75 mM NaCl. The addition of p55 to the reaction enhanced the primer extension both in the presence and absence of NaCl as complete extension of substrate was observed (Fig. 6, compare lanes 3  and 4 to lanes 5 and 6). G848S p140 extended only a few nucleotides (Fig. 6, lanes 7 and 8), and the addition of p55 stimulated   the polymerase in a salt-dependent manner (Fig. 6, compare lanes 7 and 8 to lanes 9 and 10). As expected, the lengths of these end-labeled products were proportional to overall polymerase activity. The T851A p140 extended the primer efficiently in the presence of NaCl (Fig. 6, compare lanes 11 and  12); however, in the presence of p55 the primer extension ability of the T851A p140 was moderately inhibited, and the activity of the mutant protein was rescued with the addition of 75 mM NaCl (Fig. 6, compare lane 11 to lane 13 and lane 12 to lane  14). The R852C p140 had complete primer extension in the presence of p55 in a salt-dependent fashion (Fig. 6, lanes  15-18). The primer extension ability of the R853Q enzyme was most drastically affected, as it could not extend more than seven bases even in the presence of salt and p55 (Fig. 6, lanes 19 -22). Both Q879H and T885S p140 enzymes generated high molecular weight products (Fig. 6, lanes 23 and 27). These reactions were mildly inhibited by salt (Fig. 6, lanes 24 and 28), and the p55 rescued the salt inhibition (Fig. 6, lanes 26 and 30), which is characteristic of the WT p140 enzyme (4). These assays revealed functional interaction of the p55 accessory subunit with all six mutant variants of the p140 catalytic subunit.
The true processivity of each mutant protein was studied under identical conditions, albeit in the presence of a DNA trap that binds the polymerase once it has extended the end-labeled primer-template substrate and dissociated. This single binding assay revealed that the extended primers had similar lengths to those in the primer extension assay, although the overall quantity of extended primers was significantly reduced both in the presence and absence of p55 (data not shown).
Kinetic Parameters of Mutant Proteins Reveal Catalytic Defects-Steady state kinetic measurements were performed on the WT and mutant forms of p140 using poly(dA)oligo(dT) [12][13][14][15][16][17][18] as substrate with varying concentrations of dTTP. On this substrate the WT enzyme had a K m (dTTP) ϭ 11.4 M and a k cat ϭ 13 s Ϫ1 . Steady state assumptions allow the overall efficiency of each enzyme on a homopolymeric substrate to be estimated as k cat /K m (dTTP), a parameter comparable with the pre-steady state indicator of enzymatic efficiency k pol /K d (dTTP) (22,28). Furthermore, with saturating primertemplate concentrations the ratio of k cat /K m (dTTP) does not change upon restriction of DNA synthesis (29), and k cat / K m (dTTP) remains a valid parameter for comparing mutant derivatives of pol ␥. This analysis revealed that the catalytic efficiencies (k cat /K m ) were severely compromised in the G848S, T851A, R852C, and R853Q mutant enzymes compared with the WT enzyme. These results translated to less than 1% of WT activity for the four proteins with mutations in the thumb domain (Table 2). However, the Q879H and T885S p140s displayed 46 and 86% of WT enzyme activity, respectively, suggesting that mutations in the palm subdomain can retain activity close to WT values ( Table 2).
To better understand the role of the p55 accessory subunit on the WT and mutant p140 catalytic subunits, the kinetic analysis was also performed with reconstituted WT and mutant holoenzymes (17,24). This analysis revealed that the addition of p55 had a modest effect on the WT enzyme activity (k cat ϭ 4.8 s Ϫ1 and K m ϭ 4.4 M). Including p55 only slightly enhanced the apparent catalytic efficiencies of the G848S and R853Q enzymes, whereas it showed a substantial stimulation with the R852C mutant enzyme. The addition of p55 had only a modest effect on the catalytic efficiency (k cat /K m ) of the Q879H and T885S p140 enzymes (Table 2).
Errors in Nucleotide Selection Are Not Found in These p140 Mutant Proteins-Rearrangement of mtDNA after replication errors can lead to deletion or depletion of mtDNA, and depletion of mtDNA is a hallmark of Alpers syndrome and myocerebrohepatopathy. Accordingly, we evaluated the effect of these six Alpers mutations on the fidelity of replication using a single  6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, and 30). Activity was measured at 0 mM NaCl (odd-numbered lanes) and at 75 mM NaCl (even-numbered lanes). Lanes 1 and 2 had no enzyme. The arrow indicates the position of the unextended 35-mer primer. nucleotide misincorporation assay. This gel-based assay measured the relative efficiencies of the WT and mutant p140 enzymes to incorporate the correct (dTTP) or incorrect (dATP, dCTP, and dGTP) nucleotide on a 22/40-mer primer-template oligonucleotide substrate. The steady state kinetic constants (K m and k cat ) for inserting the correct and incorrect nucleotides were determined by nonlinear curve fitting. The error frequencies (f) for the WT and mutant p140 enzymes were derived by dividing the k cat /K m values for incorrect nucleotide incorporation by the k cat /K m values for correct nucleotide incorporation (Table 3). Finally, the error frequency (f) of the WT enzyme was normalized to an arbitrary value of 1, and the error frequencies of the mutant enzymes relative to the WT p140 enzyme were calculated by dividing f MUTANT over f WT (Table 3). The results from this analysis revealed that all six mutant p140 proteins barely had an effect on the fidelity of the enzyme as judged from their f MUTANT /f WT values ( Table 3), suggesting that the misinsertion fidelity of these mutant enzymes has an insignificant role in mitochondrial disease.

DISCUSSION
Of the more than 150 disease mutations in the POLG gene, we have previously focused on adPEO mutations and other common disease mutations (16,17,24). Nearly all of the adPEO mutations in POLG are located in the polymerase region of pol ␥, and our study of four adPEO mutant pol ␥s revealed a wide range of polymerase activities ranging from 0.03% to nearly 30% of WT activity (24). Biochemical analysis of two of the common POLG disease mutations, A467T and W748S, revealed similar defects in polymerase activity but also indicated defective subunit association for the A467T p140 and significantly reduced DNA binding by the W748S p140 (16,17). For the current report we noted a cluster of mutations associated with Alpers disease and other early mtDNA depletion syndromes that was located in a unique sequence within the putative thumb domain and early palm domain of the polymerase active site. We have analyzed the biochemical consequences of six of these mutations to begin to explain the underlying molecular mechanisms leading to mtDNA depletion. This study expands the repertoire of biochemically characterized pol ␥ enzymes that harbor disease alterations (for a complete review, see Ref. 9).
Gly-848 and Arg-852 Are Located at the Face of the Thumb Helix That Interacts with DNA-The DNA polymerase activities of the G848S and R852C p140 proteins were much less than 1%, which is consistent with mtDNA depletions observed in Alpers patients with these two mutations. After A467T and W748S, G848S is the third most common mutation found in POLG, and G848S is associated not only with Alpers syndrome but also with ataxia-neuropathy syndromes, MELAS, PEO, and Leigh syndrome (14,15). Both human genetic data (tools.niehs. nih.gov/polg) and yeast data on the equivalent G651S mutation in Mip1 (30) indicate that the G848S mutation is a recessive mutation. Presentation of symptoms and progression of disease appear to depend, in part, on the identity of the mutation in the other allele (Table 1). Our biochemical analysis indicated that G848S as well as R852C pol ␥ retained less than 1% that of WT polymerase activity, but the structure of these proteins remained intact as judged by predictable chromatographic behavior during purification, CD analysis, and interaction with the accessory subunit. The equivalent change in the analogous position in the yeast Mip1 protein, G651S, causes a drastic increase in petite frequency and point mutations in mtDNA (30). One significant difference between this mutant protein and the others in this study was the 5-fold increase in K d for DNA binding. The Gly-848 residue is expected to be on the same face of an ␣-helix with Arg-852 in the thumb subdomain (Fig. 4B). Because alterations in either residue affected DNA binding, we propose that this side of the ␣-helix interacts with the DNA. Available tertiary structures of family A polymerases show interaction of the thumb domain with the minor grove of double-stranded DNA (21,31).
T851A and R853Q pol ␥ Mutations-Mutation at Thr-851 and Arg-853 also caused a substantial decrease in polymerase activity, consistent with a mtDNA depletion phenotype, but these two proteins did not exhibit altered affinity for DNA. Alanine-scanning mutagenesis of the thumb ␣-helixH of HIV-1 reverse transcriptase showed that V261A and L264A substitutions inactivated the enzyme although neither side chain on the ␣-helixH was facing the DNA substrate (32). In the absence of structural information for the human pol ␥, the reason for this polymerase defect by the T851A and R853Q mutations is unclear.
We were surprised by the profound biochemical defect displayed by the R853Q substituted pol ␥, as mutation of this same codon to encode a tryptophan residue is associated with much later age of onset POLG diseases. The R853W mutation is associated with autosomal recessive PEO when found in trans with P587L (33), and compound heterozygotes pairing R853W in trans with G737R can cause parkinsonism in the absence of PEO (34). The R853Q mutation in trans with T251I-P587L is associated with myocerebrohepatopathy in a patient that presented at 2-3 months of age. The allelic T251I-P587L mutational pair is recessive and has been found in PEO and Alpers syndrome with G848S. The absence of significant polymerase activity displayed by the R853Q pol ␥ is consistent with mtDNA depletion in the patients and helps to explain the early childhood myocerebrohepatopathy.
Q879H and T885S pol ␥ Mutations-Mutation of the moderately conserved Q879 and T885 residues only caused moder-

Misinsertion fidelity of DNA synthesis by the Alpers syndrome p140 mutants
The kinetic parameters (K m and k cat ) for incorporating a single correct (dTTP) or incorrect (dATP, dCTP, and dGTP) nucleotide onto a 3Ј-terminal matched primertemplate were measured for the WT and mutant p140 enzymes in the presence of the p55 accessory subunit as described under "Experimental Procedures." The error frequencies were then determined from the ratio f ϭ (k cat / K m ) incorrect nucleotide/ (k cat /K m ) correct nucleotide. The ratio of error frequencies for mutant enzymes relative to WT is expressed as f MUT /f WT . ate biochemical defects in pol ␥, and these defects may be insufficient to explain the severe Alpers syndrome phenotype. A closer examination of the genetics of the patient harboring the Q879H and T885S mutations offers clues to their roles in mitochondrial disease (Table 1). These two mutations were identified in a compound heterozygous patient harboring the T885S mutation along with the A467T mutation on one allele together with the Q879H and E1143G mutations on the opposite allele (35). We previously documented that the A467T p140 retains only ϳ4% polymerase activity and has impaired physical and functional interaction with the p55 accessory subunit (16). The E1143G mutation is categorized as a single nucleotide polymorphism and is found in ϳ4% of most population groups (tools. niehs.nih.gov/polg). However, we and others have documented that E1143G alters the stability and performance of pol ␥. For example, the disease mutation W748S is generally found in cis with the E1143G SNP, and we have shown that the E1143G substitution modulates both the stability and the activity of pol ␥ harboring the W748S alteration (17). Also, introduction of the E900G substitution in the yeast MIP1 gene (homologous to the E1143G substitution in human POLG) increases mtDNA mutagenesis and petite frequency at elevated temperatures, indicating a possible change of conformation relative to the WT enzyme (30). Similarly, the E1143G mutation may modulate the effects of the Q879H mutation in the Q879H-E1143G allele. Nevertheless, we attribute the majority of the deleterious effects in this patient to the A467T mutation in the A467T-T885S allele.

Enzyme
Recessive Versus Dominant POLG Mutations-Attempts to correlate a given POLG mutation with its mode of inheritance, the severity of the induced clinical phenotype, and the biochemical defects conferred on the mutant pol ␥ protein are complex but instructive. Many mutations following a dominant mode of inheritance also appear to show dominant negative biochemical behavior in vitro. For example, several POLG mutations co-segregate with the adult-onset adPEO and encode enzymes with amino acid substitutions in polymerase motif B of pol ␥. These proteins exhibited reduced DNA polymerase activity and low nucleotide selectivity in vitro, and the observed biochemical defects were generally proportional to the severity of clinical symptoms (24). One simple interpretation of this trend is that heterozygous adPEO mutations create a competition for the mtDNA replication fork between a dysfunctional and WT pol ␥. Because both copies of POLG are expressed in human cells, and mono-allelic expression of a single WT copy of POLG is sufficient to avoid disease (36), we presume that pol ␥ produced from an adPEO allele actively interferes with mtDNA replication in vivo. By extension, this model predicts that recessive missense mutations in POLG produce dysfunctional polymerases that minimally interfere with ongoing mtDNA replication catalyzed by WT pol ␥. For example, the recessive A467T mutation of POLG produces an inefficient enzyme with reduced DNA binding and defective association with the pol ␥ accessory subunit (16). This prediction is supported by the observation that a number of mitochondrial disorders result when A467T is heterozygous with other POLG mutations, and the clinical severity is largely determined by the nature of the mutation in the other POLG allele (14). Biochem-ical analysis of six recessive Alpers mutations in the current study presents challenges to this simple model. The early age of onset and the devastating severity of Alpers disease suggest profound biochemical defects in pol ␥ enzymes derived from POLG alleles with mutations linked to Alpers. Indeed, all four proteins with amino acid substitutions in the putative thumb subdomain were found to be non-functional. Like A467T pol ␥, the G848S and R852C proteins also displayed a significant reduction in DNA binding. Although the recessive mode of inheritance implies that the mutant proteins are non-competitive at the replication fork, these mutations appear to be more complex than simple loss of function mutations that predispose heterozygous individuals to mitochondrial disorders, because the clinical phenotype is not entirely controlled by the other allele. For example, A467T POLG in trans with R627Q POLG can result in ataxia-neuropathy (37), whereas compound heterozygotes bearing G848S POLG in trans with R627Q POLG can present with MELAS (38). Does the residual DNA polymerase activity of A467T pol ␥ contribute to a milder phenotype? Although the Alpers proteins may not directly compete with WT p140, they may compete indirectly through interaction with other proteins at the mtDNA replication fork. Unlike A467T p140, each of the Alpers proteins retains physical and functional interaction with the pol ␥ accessory subunit. Does the enhanced DNA binding conferred by the accessory subunit reduce dissociation of mutant pol ␥ from mtDNA, thereby interfering with the ability of a WT enzyme to complete replication? Clearly, knowledge of the complex interactions of all the proteins in the mitochondrial replisome is needed to refine the model to address these more difficult questions.
Thumb Subdomains in Other DNA Polymerases-In prototypical family A polymerases, such as the E. coli pol I, the thumb subdomain consists of two flexible ␣-helices of ϳ50 amino acids that interact with the incoming primer-template (31). Consistent with these findings, deletion of 24 amino acids in the Klenow fragment corresponding to part of the tip of the thumb subdomain causes a 100-fold reduction in DNA binding affinity, reduces processive synthesis, and increases the rate of frameshift errors (39). G848S pol ␥ (and to a lesser extent R852C pol ␥) also displayed a decreased DNA binding affinity. None of the mutant proteins studied displayed a significant decrease in misinsertion fidelity. Consistent with this observation, alanine scanning mutagenesis of conserved residues in the tip of the Klenow thumb also did not display an increase in misinsertion (point mutations) or frameshift errors (40). As in most DNA polymerases, the thumb subdomain is structurally conserved in HIV-1 reverse transcriptase (41). Alanine scanning mutagenesis of amino acid residues in the HIV-1 reverse transcriptase thumb structure revealed that mutations in three residues, Gln-258, Gly-262, and Trp-266, in ␣-helixH had a significant decrease in primer-template binding, base substitutions, and frameshift fidelity (32,42). Interestingly, the periodicity of these residues in the ␣-helixH shows that they are all on the same helical face that interacts with minor groove of the DNA (32,42). Although none of our Alpers mutant p140 enzymes displayed any significant change in fidelity, based on the defect in DNA binding we predict that the Gly-848 and