Human Mitochondrial DNA Polymerase Holoenzyme: Reconstitution and Characterization
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- 作者：Allison A. Johnson ; Yu-chih Tsai ; Steven W. Graves ; and Kenneth A. Johnson
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：February 22, 2000
- 年：2000
- 卷：39
- 期：7
- 页码：1702 - 1708
- 全文大小：174K
- 年卷期：v.39,no.7(February 22, 2000)
- ISSN：1520-4995
文摘
We have reconstituted the holoenzyme of the human mitochondrial DNA polymerase fromcloned and overexpressed catalytic and accessory subunits. We have examined the polymerization activityof the catalytic subunit alone and of the holoenzyme to establish the function of the accessory subunit inthis two subunit enzyme. The accessory subunit associates with the catalytic subunit with a dissociationconstant of 35 ± 16 nM as measured by the concentration dependence of its effect in stimulating maximalDNA binding and polymerization. At saturating concentrations, the accessory subunit contributes to everykinetic parameter examined to facilitate tighter binding of DNA and nucleotide and faster replication.The accessory protein makes the DNA binding 3.5-fold tighter (Kd of 9.9 ± 2.1 nM compared to 39 ±10 nM for the catalytic subunit alone) without significantly affecting the DNA dissociation rate (0.02 ±0.001 compared to 0.03 ± 0.001 s-1). The ground-state nucleotide binding is improved from 4.7 ± 2.0to 0.78 ± 0.065 
M, and the maximum DNA polymerization rate is increased from 8.7 ± 1.1 to 45 ± 1s-1 by the addition of the accessory protein. This leads to an increase in processivity from an estimated290 ± 46 to 2250 ± 162. Although the accessory protein has been described as a "processivity factor"because of its effect on the ratio of rate constants defining processivity, this terminology falls short ofadequately describing the profound effects of the small subunit on nucleotide-binding and incorporationcatalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with acomprehensive analysis of the structural and mechanistic determinants of enzyme specificity that governtoxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.
M, and the maximum DNA polymerization rate is increased from 8.7 ± 1.1 to 45 ± 1s-1 by the addition of the accessory protein. This leads to an increase in processivity from an estimated290 ± 46 to 2250 ± 162. Although the accessory protein has been described as a "processivity factor"because of its effect on the ratio of rate constants defining processivity, this terminology falls short ofadequately describing the profound effects of the small subunit on nucleotide-binding and incorporationcatalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with acomprehensive analysis of the structural and mechanistic determinants of enzyme specificity that governtoxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.