Mitochondrial DNA is replicated and repaired by DNA polymerase γ (pol γ). In mammalian cells, pol γ is a heterotrimer composed of a large 140 kDa catalytic subunit associated with two 55 kDa accessory proteins as a homodimer (Graziewicz et al., 2006
; Kaguni, 2004
). The catalytic subunit contains the DNA polymerase active site, a 3′-5′ exonuclease for proofreading, and an intrinsic 5′-dRP lyase activity needed for single nucleotide base excision repair. The p55 homodimer accessory subunit binds asymmetrically to the catalytic subunit and imparts high processivity on the complex by increasing its affinity to DNA (Lee et al., 2009
; Lim et al., 1999
). The catalytic subunit is encoded by a 23 exon gene on chromosome 15, 15q25 (Ropp and Copeland, 1996
). In 2001, the first set of POLG
mutations were identified in three families with progressive external opthalmoplegia, (PEO) (Van Goethem et al., 2001
). This was followed by several publications reporting signs of PEO and ataxia-neuropathy with mtDNA deletions. In 2004, POLG
mutations were identified in two unrelated pedigrees of Alpers-Huttenlocher syndrome (AHS) that caused mtDNA depletion (Naviaux and Nguyen, 2004
To date more than 160 pathogenic mutations have been reported in the POLG
gene as well as several single nucleotide polymorphisms (Stumpf and Copeland, 2011
) (see http://tools.niehs.nih.gov/polg/
). These mutations are associated with at least six varied disease states that affect humans starting from soon after birth until late in life (Saneto and Naviaux, 2010
; Wong et al., 2008
). Early infant and childhood presentations of POLG
related disorders cause mtDNA depletion and include AHS and myocerebrohepatopathy spectrum disorder with recessive inheritance. Later onset (after 10 yrs of age) POLG
mutations usually cause mtDNA deletions and include ataxia-neuropathy, myoclonus epilepsy myopathy sensory ataxia, and autosomal recessive and dominant forms of PEO.
Biochemical analysis of the recombinant human pol γ harboring disease substitutions has been a useful tool for understanding the consequence and mechanism of POLG
-related disorders. For example, analysis of four autosomal dominant PEO mutations, G923D, R943H, Y955C, and A957S, demonstrated varied degree of defect, but correlated with the severity of the disease as well as age of onset (Graziewicz et al., 2004
). Analysis of pol γ with most common disease mutation, A467T, indicates that the Ala to Thr substitution imparts instability, decline of polymerase activity, and a defect of interacting with the p55 accessory subunit (Chan et al., 2005
). More recently, biochemical analysis of four AHS mutations in highly conserved amino acids in the thumb subdomain showed nearly total loss of polymerase activity, concordant with the early presentation and severity of AHS in combination with other POLG
mutations (Kasiviswanathan et al., 2009
One such mutation that has been frequently reported in a variety of conditions and age of presentation is the G517V mutation. The G517V mutation was originally reported in a large study of patients with POLG
mutations (Horvath et al., 2006
). In this study, the authors identified the G517V mutation as an autosomal dominant mutation in three individuals over three generations in one family with neuropathy or epilepsy. The mutation was also found in 1 control subject out of 672 alleles from German control subjects.
The G517V mutation has since been identified in many patients with varying symptoms. Sarzi et al. reported the G517V heterozygous mutation in a patient with psychomotor retardation that died from Leigh syndrome (Sarzi et al., 2007
). Her father also carried this mutation but was asymptomatic. Wong et al. found the G517V mutation as a single heterozygous mutation in 5 unrelated subjects (Wong et al., 2008
). These patients displayed variable disease presentation including Leigh-like signs, neuropathy, myopathy, sideroblastic anemia and Pearson syndrome to Kearns-Sayre syndrome. The age of onset in these five patients also varied from 2-16 years of age. Furthermore, the G517V mutation was found as a compound heterozygote in two 1-year old patients, one with D1196N with myopathy and ragged red fibers and the second patient with a R1128H mutation that suffered from microcephaly. However, neither patient demonstrated the typical signs of early POLG
-related disorders like AHS.
In 2009, Blok et al. reported four mitochondrial disease patients with the single heterozygous G517V mutation in POLG
(Blok et al., 2009
). The patients ranged in age from 4-40 years and displayed signs of chronic PEO with ataxia and hypotonia (in the 4 yr old), a single unexplained attack of status epilepticus in a 10 yr old, a 34 yr old with cerebellar ataxia, dystonia and mild retardation, and a 40 year old with mental retardation and chorea while giving birth (Blok et al., 2009
The G517V POLG
mutation was also found as a single heterozygous mutation in a 8.5 month old boy with electron transport deficiency and his 7 year old half sister (Burusnukul and de los Reyes, 2009
). The 8.5 month old boy was examined for myoclonic jerks, developmental delay, and epilepsy, and he was found to have impaired fasting glucose tolerance, decreased complex 1 activity (9% of normal), and deceased complex 3 activity (32% of normal). No mitochondrial DNA point mutations or deletions were detected. His 7 year old half sister had symptoms of seizures, migraines and was developmentally delayed.
In another infant case, a 6 month old boy was admitted with myoclonic epilepsy that resembled mitochondrial disease (Bolszak et al., 2009
). The G517V mutation was found in this patient in trans with the R722H POLG
mutation. The boy was treated with various anti –seizure drugs including valproate. At age 6 and later the brain MRI was normal. The 46 year old mother that carried the G517V mutation was asymptomatic.
Hopkins et al. identified the G517V heterozygous mutation in twin sisters, their sister and mother in a proband that developed type I diabetes, adrenal insufficiency, hypothyroidism and psychiatric problems and concluded an autosomal dominance mode of inheritance (Hopkins et al., 2010
). Finally, Schulte et al identified the G517V mutation in a single 44 yr old patient with ataxia plus ophthalmoplegia without neuropathy (Schulte et al., 2009
Thus, the G517V mutation has been reported as a single heterozygous POLG mutation in patients ranging in age from 8 months to 50 years. As a compound heterozygous mutation with other POLG mutations the patients have been around 1 year of age but the symptoms were very different and one patient apparently recovered. To gain a better understanding of this mutation and determine if the Gly to Val mutation elicits a change in pol γ’s enzymatic activity, we biochemically characterized the recombinant wild type pol γ and G517V variant. We found that the G517V pol γ behaved similarly to the wild type enzyme suggesting that the mutation has no pathological consequence.