Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations
Introduction
Of the 16 DNA polymerases in the eukaryotic cell, only pol γ is known to function in animal mitochondria [1], [2], [3]. Thus, pol γ is absolutely essential for mitochondrial DNA replication and repair. The holoenzyme of pol γ consists of a catalytic subunit (encoded by POLG at chromosomal locus 15q25) and a dimeric form of its accessory subunit (encoded by POLG2 at chromosomal locus 17q24.1). The catalytic subunit is a 140 kDa enzyme (p140) that contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain and has DNA polymerase, 3′ → 5′ exonuclease and 5′ dRP lyase activities [4]. The accessory subunit is a 55 kDa protein (p55) required for tight DNA binding and processive DNA synthesis [5]. The pol γ holoenzyme functions in conjunction with the mitochondrial DNA helicase (Twinkle) and the mitochondrial SSB to form the minimal replication apparatus [6].
The POLG gene is one of several nuclear genes that is associated with mitochondrial DNA depletion or deletion disorders. To date, more than 150 disease mutations have been identified in the POLG gene and an up-to-date mutation database can be found at http://tools.niehs.nih.gov/polg/, which shows these mutations equally distributed over the length of the protein. Disorders associated with POLG mutations include (1) myocerebrohepatopathy spectrum disorder (MCHS), (2) Alpers syndrome, (3) ataxia neuropathy spectrum disorder (ANS), (4) myoclonus epilepsy myopathy sensory ataxia (MEMSA), (5) autosomal recessive progressive external ophthalmoplegia (arPEO), and (6) autosomal dominant progressive external ophthalmoplegia (adPEO) [7]. Also, alteration of the (CAG)10 repeat in the 2nd exon of POLG has been implicated in male infertility [8].
A summary of previous structure–function studies in pol γ that were performed to address disease mutations has recently been published [9]. Among all disease mutations, A467T mutation is the most common POLG mutation and has been found to be associated with all of the disease symptoms mentioned above. Previous studies have shown that the A467T pol γ possesses only 4% of the wild-type DNA polymerase activity and is compromised for its ability to interact with the p55 accessory subunit [10]. Another mutation, W748S, which has nearly always been found to be in cis with the E1143G mutation, is a frequent cause of ataxia-neuropathy syndrome [11]. The E1143G substitution, a single nucleotide polymorphism (SNP), is found in 4% of European populations. The W748S mutation has intrinsic lower polymerase activity as well as a demonstrated lower affinity for DNA [12]. We have found that the E1143G SNP can modulate the deleterious effect of the W748S mutation [12]. This finding raises the possibility that other SNPs could potentially affect POLG enzymatic activity.
Four adPEO mutations, G923D, R943H, Y955C and A957S that are found in and around motif B in the active site of the polymerase were characterized biochemically [13]. Two of the substitutions (R943H and Y955C), change side chains that interact with the incoming dNTP, and pol γ with either of these substitutions retains less than 1% of the wild-type polymerase activity and displays a severe decrease in processivity. The significant stalling of DNA synthesis and extremely low catalytic activities of both enzymes are the two most likely causes of the severe clinical presentation in R943H and Y955C heterozygotes [13]. The substitution of Tyr955 to cysteine also increases nucleotide misinsertion replication errors 10–100-fold in the absence of exonucleolytic proofreading [14]. Lately, we also characterized six Alpers mutations, four of which are highly conserved and located in the thumb subdomain of the polymerase portion of the enzyme (G848S, T851A, R852C and R853Q) [15]. Purified recombinant pol γ proteins containing these point mutations exhibited less than 1% WT enzyme activity levels in addition to reduced DNA binding exhibited by the G848S and R852C enzymes [15]. For the majority of the disease substitutions that have been studied in vitro, the biochemical defects correlate with the severity and age of onset found in patients [9]. Further analysis of disease substitutions as well as structural analysis should aid in the continued understanding of disease mutations in the POLG gene.
Recently, we described a summary of assays used to characterize the pol γ catalytic subunit [16]. In this methods review, we have updated those methods and include summaries of the purification schemes required to purify the pol γ catalytic subunit (p140) and the p55 accessory subunit. Furthermore, as an example of how to use these assays to characterize a disease mutation, we include analysis for the R964C mutant pol γ that has been recently described [17].
Section snippets
Construction of human DNA polymerase γ mutants
QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce mutations in the cDNA encoding the exonuclease deficient (Exo−) pol γ (POLG). The pol γ Exo− background was created by mutating two crucial amino acids in the exonuclease domain (D198A/E200A) to abolish the 3′ → 5′ exonuclease activity that may interfere with biochemical assays involving nucleic acids [18]. This POLG cDNA without its mitochondrial targeting sequence was cloned into the pVL1393 baculovirus transfer vector
Acknowledgments
The authors would like to thank Drs. Deepti Dwivedi and Rajendra Prasad for critical reading of this manuscript. This work was supported by the NIEHS Intramural Research Program.
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