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Characterization of a DNA primase from rat liver mitochondria
Abstract:
A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.
Insights
Mitochondrial DNA primase synthesizes mixed RNA-DNA primers, crucial for DNA replication initiation. This enzyme
Area of Science:
- Mitochondrial DNA replication
- Enzymology
- Biochemistry
Background:
- DNA primase is essential for initiating DNA synthesis by creating RNA primers.
- Mitochondrial DNA polymerase gamma and mtRNA polymerase are distinct enzymes involved in mitochondrial nucleic acid metabolism.
- Understanding the precise function and regulation of mitochondrial DNA primase is key to comprehending mitochondrial DNA replication fidelity.
Purpose of the Study:
- To partially purify and characterize rat liver mitochondrial DNA primase.
- To differentiate DNA primase activity from DNA polymerase gamma and mtRNA polymerase.
- To investigate the role of dATP in regulating primase activity and primer synthesis.
Main Methods:
- Partial purification of DNA primase from rat liver mitochondria using heparin-agarose chromatography.
- Enzyme activity assays to synthesize polynucleotide products using poly(dT) template.
- Analysis of reaction products and enzyme kinetics under varying conditions (pH, cations, ATP, dATP concentrations).
Main Results:
- Mitochondrial DNA primase was separated from DNA polymerase gamma and mtRNA polymerase.
- Primase synthesized mixed oligo(A)-oligo(dA) products on a poly(dT) template.
- dATP inhibited AMP incorporation by reducing new chain initiations, but did not fully convert primase to DNA synthesis mode.
Conclusions:
- The data support a model where mitochondrial DNA primase synthesizes mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide primers.
- The formation of the RNA-DNA junction during primer synthesis is an intrinsic function of primase.
- Mitochondrial DNA primase plays a critical role in initiating DNA replication, with its activity potentially regulated by dATP levels.