Abstract
Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol gamma), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol gamma 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 gamma 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 gamma 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 gamma and examines the consequences of mitochondrial disease mutations in this region.
Highlights
Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase, cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome
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
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]
Summary
Construction of Substituted p140 Proteins—Mutations in the cDNA encoding the exonuclease-deficient (ExoϪ) pol ␥ (POLG) were generated using the QuikChange site-directed mutagenesis kit (Stratagene) with the pQVSL11.4 baculoviral transfer vector encoding p140 ExoϪ without its mitochondrial targeting sequence [22] as template. For steady state kinetic values the same assay was performed in the presence and absence of the p55 accessory subunit using poly(dA)-oligo(dT) as substrate, with reactions containing 25 mM NaCl as previously described [4]. The 50-l reaction contained 50 mM HEPES-KOH (pH 8.0), 2 mM 2-mercaptoethanol, 10 mM MgCl2, 0.1 mg/ml heattreated BSA, 50 M dNTPs, 13.3 nM [␣-32P]dTTP, 5 g of the activated calf thymus DNA, and 100 ng of purified WT or mutant p140 enzyme. The 10-l reaction contained 25 mM HEPES-KOH (pH 7.6), 5 mM 2-mercaptoethanol, 5 mM MgCl2, 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. POLG mutations located in the thumb region characterized in this study, associated clinical phenotypes, and other POLG mutations found in trans
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