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Defects in mtDNA replication are the principle cause of severe, heritable metabolic disorders classified as mitochondrial diseases. In vitro analysis of the biochemical mechanisms of mtDNA replication has proven to be a powerful tool for understanding the origins of mitochondrial disease. Mitochondrial single-stranded DNA binding protein (mtSSB) is an essential component of the mtDNA replication machinery. To facilitate ongoing biochemical studies, a recombinant source of mtSSB is needed to avoid the time and expense of human tissue culture. This chapter focuses on the sub-cloning, purification, and initial functional validation of recombinant human mitochondrial single-stranded DNA binding protein. The cDNA encoding the mature form of the human mtSSB protein was amplified from a HeLa cDNA library, and recombinant human mtSSB was over-produced in E. coli. A procedure was developed to rapidly purify milligram quantities of homogenous, nuclease-free mtSSB that avoids DNA-cellulose chromatography. We show that, similar to E. coli SSB, human mtSSB assembles into a tetramer and binds single-stranded oligonucleotides in a 4-to-1 protein:oligonucleotide molar ratio.
Human mitochondria possess a circular, double-stranded DNA chromosome (16,569 bp) that is indispensible for the healthy growth of cells. Mitochondrial DNA (mtDNA) encodes 2 rRNAs, 22 tRNAs and 13 mRNAs essential for electron transport and oxidative phosphorylation. Mutation of mtDNA causes a wide spectrum of neuromuscular degenerative diseases affecting many different tissues, and defects in mtDNA replication are the principle cause of metabolic disorders classified as “mitochondrial diseases”. All of the factors required for replication and repair of the mitochondrial genome are known to be encoded by the nucleus, imported into mitochondria as pre-proteins, and proteolytically processed into their mature forms. Efforts to define the genetics and the biochemical mechanisms by which cells maintain the integrity of their mtDNA are essential to understanding the origins of mitochondrial diseases.
Mitochondrial single-stranded DNA binding protein (mtSSB) was discovered in an analysis of protein-mtDNA complexes derived from rat liver mitochondria that had been lysed with SDS, which revealed nucleoprotein fibrils within the single-stranded portions of both stable and expanding D-loops in replicative intermediates of rat liver mtDNA (1). Current models of mtDNA replication predict replication intermediates that contain large regions of single-stranded DNA, and the abundant presence of 16 kDa mtSSB in these nucleoprotein fibrils strongly suggests that the mtSSB protein is an essential component of the mtDNA replication machinery. Also, native mtSSBs isolated from Drosophila, Xenopus, and HeLa have been reported to stimulate DNA polymerase γ from these organisms, dependent on the substrate utilized in vitro (2–4). In addition, mtSSB has been shown in vitro to relieve replication stalling by Xenopus laevis pol γ within dT-rich template DNA sequences (5). More recently, human mtSSB has been shown to stimulate activity of the mtDNA helicase, and in vitro mtSSB is part of a minimal replisome complex along with the helicase, DNA polymerase γ and its accessory protein (6,7). Mutations in the nuclear genes encoding mtDNA replication components, specifically the DNA polymerase γ (POLG and POLG2) and the helicase (PEO1), have been clearly linked with mitochondrial disease (8,9). The gene for human mtSSB has been cloned, and the protein has been shown to be homologous to E. coli SSB (10). Although mtSSB has not yet been identified as a disease locus in humans, it is essential in yeast (11), and participation in mtDNA replication makes mtSSB an obvious candidate locus for mitochondrial disease in humans.
Existing procedures for the purification of native mitochondrial or E. coli SSBs consistently utilize single-stranded DNA-cellulose chromatography as a primary purification step (2,12,13). In our hands, recombinant mtSSB prepared from E. coli BL21(DE3) lysates by such protocols contained unacceptably high levels of nuclease activity that could not be resolved by additional chromatography on Affi-Gel Blue (14) or hydroxylapatite. Previous reports describing the purification of recombinant human mtSSB rely on animal cell culture as the source of protein and/or affinity tags to aid purification (6,15,16). Here, we describe over-expression in E. coli of the mature form of human mtSSB without an affinity tag and without the mitochondrial targeting sequence, and a procedure for rapid purification of milligram quantities of homogenous, nuclease-free mtSSB that does not use DNA-cellulose chromatography is presented.
Utilizing oligonucleotide primers designed from the published nucleotide sequence of the human gene (10), the cDNA for the human mtSSB was amplified from a HeLa cDNA library. Expression of the full-length human cDNA (aa 1–148, MW 17249) in E. coli (JM105) from a pQE9 expression vector (Qiagen) generated recombinant protein that was almost entirely insoluble (data not shown). Similarly, Li and Williams demonstrated that alteration of the N-terminal amino acid sequence of murine mtSSB, such as by insertion of an affinity-tag to aid protein purification or even by the simple retention of the mitochondrial targeting presequence, adversely affected DNA binding and/or tetramerization of recombinant murine mtSSB (15). Accordingly, we deleted the mitochondrial targeting sequence identified by Tiranti (10) and transferred the cDNA encoding the mature form (aa 17–148, MW 15316) into the pET21a expression vector (Novagen). The resulting plasmid, pET21aHmtSSB, was used to transform E. coli JM105(DE3) to ampicillin resistance. (see Notes 1 and 2). Ammonium sulfate was from Invitrogen, Carlsbad, CA. Ampicillin, IPTG, myo-inositol, CHES were obtained from Sigma. E. coli single-stranded DNA binding protein was purchased from United States Biochemical. Synthetic oligonucleotides were from Oligos Etc. (Wilsonville, OR).
E. coli JM105(DE3) bearing a pET21a vector encoding the mature form of human mitochondrial SSB was treated with IPTG to induce gene expression and lysed by sonication. Cleared lysates prepared from 1 liter cultures were applied to Affi-Gel Blue resin, extensively washed, and mtSSB was eluted with 0.5 M KSCN before fractional precipitation with 10 – 35% (saturation) ammonium sulfate. Following dialysis, the protein fraction that did not bind MonoS was adjusted to pH 9.3 and applied to a MonoQ FPLC column. Homogenous mtSSB was eluted from MonoQ with a linear salt gradient. Typical yield from a 1 L culture was 4 – 6 mg homogenous mtSSB (see Table 1). Detailed procedures are listed below.
Validation experiments were performed both to verify the purity of recombinant, mature human mtSSB as prepared by this method, as well as to confirm the ability of the purified protein to form homotetramers and bind to single-stranded DNA.
Established procedures for the purification of prokaryotic and mtSSB's often rely upon single-stranded DNA-cellulose chromatography as a terminal purification step. To avoid the possibility of DNA contaminating the mtSSB preparation, as well as to minimize the chances of enriching for other DNA metabolizing enzymes due to their intrinsic affinity to DNA, we sought to develop a rapid protocol for purifying recombinant mtSSB with a high yield that does not utilize DNA affinity chromatography. Samples taken throughout the purification were analyzed by SDS-PAGE (see Fig. 1). The high specificity of Affi-Gel Blue rendered the mtSSB >90% pure after the first chromatographic step. KSCN was removed by ammonium sulfate precipitation, and passage through a MonoS column removed many higher MW contaminants, including a contaminating nuclease activity. Although E. coli SSB and mtSSBs do not readily bind MonoS or MonoQ FPLC columns at neutral pH, MonoQ binds human mtSSB with high capacity at pH 9.5, permitting purification to homogeneity without resorting to DNA-cellulose chromatography.
Equilibrium sedimentation analysis was performed to determine the native conformation of purified, recombinant, human mtSSB. In preparation for analytical ultracentrifugation, glycerol was removed from a sample of mtSSB (Fraction V) by dialysis against a buffer containing 30 mM HEPES•KOH (pH 7.6), 2 mM 2-mercaptoethanol, 0.25 mM EDTA, 0.25 M KCl. Protein concentration was determined spectrophotometrically, as in in Subheading 3.7. Samples (100 μL) were adjusted by dilution with dialysis buffer to protein concentrations of 32.1, 16.0, and 10.7 μM mtSSB (monomers), and subjected to equilibrium sedimentation in an Optima XL-A analytical ultracentrifuge (Beckman Instruments) at 11,000 rpm at 4°C prior to obtaining absorbance profiles at 280 nm (see Fig. 2). Profiles were fit with Optima XL-A data analysis software, assuming a partial specific volume of 0.7261 cm3/g and a solvent density of 1.0128 g/cm3. The random distribution of residual absorbance values indicated that all three profiles fit very well to a model of a single species (see Fig. 2) with an average molecular weight of 62,600 ± 1760 Da. Although attempts to model monomer-dimer or monomer-tetramer equilibria were confounded by the inability to detect SSB monomers at these protein concentrations, the subunit dissociation constant is estimated to be less than 10−8 M. Because the recombinant protein has a predicted molecular weight of 15,316 Da, the measured value indicates that recombinant human mtSSB exists as a tetramer in solution, as observed previously for native and recombinant forms of mtSSB from Xenopus laevis (3,18), Drosophila melanogaster (2), and humans (13).
The ability of mtSSB tetramers to bind single-stranded DNA was confirmed by electrophoretic mobility shift assay, as described previously (19). DNA-binding by E. coli SSB, which also exists as a tetramer in solution (20), was assessed as a positive control in side-by-side reactions. Various concentrations of human mtSSB or E. coli SSB were combined in vitro with radiolabeled, single-stranded 34mer oligonucleotide, and mixtures were resolved by native PAGE (see Fig. 3, upper panel). This titration experiment indicates that 40 fmol of oligonucleotide are saturated by approximately four equivalents of either E. coli SSB monomers or mtSSB monomers, confirming the similar DNA-binding strength of each tetrameric species in vitro (see Fig. 3, lower panel).
The authors would like to thank Dr. Harvey Sage of the Department of Biochemistry at Duke University Medical Center for performing the analytical ultracentrifugation, as well as Drs. Leroy Worth (NIEHS) and Sherine Chan (NIEHS) for thoughtful comments on the manuscript. This work was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences.