Human Mitochondrial DNA Polymerase Holoenzyme: Reconstitution ...

Human Mitochondrial DNA Polymerase Holoenzyme: Reconstitution ...

Human Mitochondrial DNA Polymerase Holoenzyme: Reconstitution and Characterization Translated by: Perray Saravanane Holoenzyme Enzymes with protein and non-protein components Complete/catalytically active form of an enzyme Consists of a apoenzyme (inactive form) and its cofactor/s

Major subunits of DNA Poly. G Catalytic Polymerase function (5-3) exonuclease function (3-5) Accessory Tighter binding of DNA and nucleotide Maximum DNA polymerization rate

increases Faster replication Called a processivity factor Key areas of investigation The individual function of the accessory subunit Finding the correct cDNA (complementary DNA) in Homo Sapiens Assembling a soluble, active human mitochondrial DNA polymerase holoenzyme Containing artificial catalytic and accessory subunits

Experimental Procedures Creation of cDNA strands/Bacterial Construct ORF of 485 amino acid sequence was found by the authors for the human mitochondrial DNA poly accessory subunit. AA sequences of Hs, Xl, and Dm

were aligned using Clustal X and allowed for truncation of the sequence due to the shared homology PCR was run on the truncated sequences which were restriction digested, purified, and the bacteria was grown in broth samples (centrifuged) The bacterial samples underwent sonication and another round of

centrifugation, the supernatant was kept Chromatography methods + Western Blot Chelating sepharose chromatography was run to distinguish and purify proteins with affinities to chelating ions. Mono-S chromatography was used to allow for high resolution polishing of proteins for analysis.

Figure 2B is the result of the western blot gel with six different lanes of cell fractions. Each lane refers to a previous stage where the cell culture was further purified using a unique method. Pre-Steady/Steady State Assays (Single Nucleotide

Incorporation) Steady State rate of elongation Pre-Steady State rate of elongation Rapid

Quench Flow Instrument Catalytic and Accessory Subunit Interaction The dissociation constant for the reconstitution

of holoenzyme was observed Active Site Titration The dissociation constant for the DNAbinding to holoenzyme + concentration of active enzyme was observed Nucleotide Dissociation Constant and

Maximum Polymerization Rate Results The authors have demonstrated that the accessory subunit plays a role in : Tighter polymerase binding to the DNA

Tighter poly. Binding to the nucleotide Increased rate of nucleotide incorporation into DNA Significance In combating diseases such as AIDS and Hepatitis B, nucleoside analogues are used to decrease the binding of the DNA backbone from

occurring, however a major side of effect is causing a decrease in mitochondrial function. Through a better understanding of the complex nature of DNA poly. Gamma and its subunits, researchers can perform structure-function studies to screen new nucleoside analogues that have low toxicity levels.

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