Expression, purification, and initial kinetic characterization of the large subunit of the human mitochondrial DNA polymerase.

Abstract:

Faulty replication of the human mitochondrial genome is thought to be the cause of many diseases; moreover, the low selectivity of the mitochondrial DNA polymerase has been implicated as the cause of many side effects observed in the treatment of viral infections such as HIV. To better understand how the mitochondrial genome is replicated, we cloned a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit was recombinantly expressed and purified to near homogeneity. The purified enzyme demonstrated both polymerase and 3'-5' exonuclease activity. The purified protein was examined in single nucleotide incorporation assays, demonstrating that the enzyme had a maximum polymerization rate of 3.5 s-1 and a dissociation rate from the DNA substrate of 0.03 s-1, affording a calculated processivity of 116. The dissociation constants for the enzyme binding to DNA and nucleoside triphosphate were 39 nM and 14 microM, respectively. The 3'-5' exonuclease rate was measured at 0. 18 s-1. Though the slow rate of polymerization suggests that the large subunit of human DNA polymerase gamma may require accessory factors to increase its processivity of polymerization, the kinetic parameters indicate that the large subunit of DNA polymerase gamma could replicate the mitochondrial genome in a physiologically relevant time frame. This study provides the initial characterization of the large subunit of DNA polymerase gamma and establishes the baseline for examination of the effects of accessory proteins such as the putative small subunit.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
HIV RT Graves SW1998 Processivity 200bp
HIV RT Graves SW1998 Vmax 30 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
HIV RT Graves SW1998 Kd 4.7nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
HIV RT Graves SW1998 Kd 14uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Klenow fragment Graves SW1998 Processivity 250bp
Klenow fragment Graves SW1998 Vmax 50 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Klenow fragment Graves SW1998 Kd 5nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Klenow fragment Graves SW1998 Kd 17uM Reaction: Nucleotide incorporation; Substrate: dNTPs
T7 Graves SW1998 Processivity 1500bp
T7 Graves SW1998 Vmax 300 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T7 Graves SW1998 Kd 18nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
T7 Graves SW1998 Kd 18uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Human Pol gamma Graves SW1998 Molecular Weight 1.38E+05 Dalton
Human Pol gamma Graves SW1998 3-5' Exonuclease (proofreading) Yes
Human Pol gamma Graves SW1998 Tagged Yes
Human Pol gamma Graves SW1998 Tag Name His
Human Pol gamma Graves SW1998 Full length or truncated Full length
Human Pol gamma Graves SW1998 Processivity 116bp
Human Pol gamma Graves SW1998 Vmax 3.5 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Human Pol gamma Graves SW1998 Kd 39nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Human Pol gamma Graves SW1998 Kd 14uM Reaction: Nucleotide incorporation; Substrate: dTTP
Human Pol gamma Graves SW1998 kcat 0.03 /second
Human Pol gamma Graves SW1998 kcat 0.18 /second Reaction: 3-5' Exonuclease; Substrate: DNA template

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