Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations

Abstract

More than 150 different point mutations in POLG, the gene encoding the human mitochondrial DNA polymerase gamma (pol gamma), cause a broad spectrum of childhood and adult onset diseases like Alpers syndrome, ataxia-neuropathy syndrome and progressive external ophthalmoplegia. These disease mutations can affect the pol gamma enzyme's properties in numerous ways, thus potentially influencing the severity of the disease. Hence, a detailed characterization of disease mutants will greatly assist researchers and clinicians to develop a clear understanding of the functional defects caused by these mutant enzymes. Experimental approaches for characterizing the wild-type (WT) and mutant pol gamma enzymes are extensively described in this manuscript. The methods start with construction and purification of the recombinant wild-type and mutant forms of pol gamma protein, followed by assays to determine its structural integrity and thermal stability. Next, the biochemical characterization of these enzymes is described in detail, which includes measuring the purified enzyme's catalytic activity, its steady-state kinetic parameters and DNA binding activity, and determining the physical and functional interaction of these pol gamma proteins with the p55 accessory subunit.

Figure 1

Purification of recombinant pol γ subunits. (A) Catalytic subunit of pol γ enzyme was purified and samples from various steps were analyzed on a 4–20% gradient SDS-PAGE gel. Lane 1, whole cell extract; lane 2, soluble lysate; lane 3, phosphocellulose P-11 flow-through; lane 4, Ni-NTA column flow-through; lane 5, Fast Desalt HR 10/10 column eluate; lanes 6–9, peak fractions eluted from Mono Q column. M, Molecular weight markers. (B) Purification of human p55 from BL21(DE3) E. coli. Samples were analyzed by 4–20% SDS-PAGE and stained with Coomassie Brilliant Blue. M, SDS-PAGE Molecular weight markers and the positions of molecular weight standards in kDa are indicated; Lane 1, E. coli whole cell extract generated by French Press; Lane 2, soluble E. coli lysate following 30,000 × g centrifugation; Lane 3, unbound proteins following batch binding of soluble lysate to Ni-NTA agarose; Lane 4, Ni-NTA agarose eluate pool; Lanes 5–11, MonoS fractions 24–30. Fractions 29 and 30 were pooled and subsequently used for biochemical analyses.

Figure 2

Secondary structure of WT and R964C pol γ proteins and their stability. (A) CD spectroscopy of WT (circles) and R964C (squares) proteins. (B) Thermal stability of WT and R964C mutant proteins. The enthalpy change during protein folding (ΔH_m), measured at 220 nm is represented by bars with standard deviations. The melting temperature (T_m) is represented by circles. The results are an average of three independent experiments.

Figure 3

DNA binding affinity of WT and R964C pol γ proteins. Representative gels showing electrophoretic mobility shift assays performed using (A) WT and (B) R964C mutant pol γ enzymes as described in Methods to estimate the K_{d (DNA)} values. Lanes 1–8 contained 50 nM substrate; Lanes 2–8 had 0, 25, 50, 75, 100, 150, 200 or 250 nM of WT (A) or R964C mutant (B) pol γ protein. S, substrate, P, protein-DNA complex.

Figure 4

Functional interaction of WT and R964C pol γ enzymes with the p55 accessory subunit. Primer extension assays were performed using WT and R964C pol γ enzymes in the presence and absence of p55 accessory subunit on singly primed M13 DNA as described in Methods. Reactions contained 2 nM substrate (all lanes), 5 nM pol γ enzymes (lanes 3–6, WT; lane 7–10, R964C), 10 nM p55 accessory subunit (lanes 5, 6, 9 and 10). 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.