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DNA replication in vitro by recombinant DNA-polymerase-alpha-primase
F Stadlbauer1, A Brueckner, C Rehfuess
1Institut für Biochemie d. LMU München, Germany.
Abstract:
DNA-polymerase-alpha--primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-alpha--primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-polymerase-alpha--primase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-alpha--primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-alpha--primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.
Insights
Researchers characterized the DNA-polymerase-alpha--primase complex, identifying subunits responsible for primase and polymerase activities. This study elucidates the complex
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- The DNA-polymerase-alpha--primase complex is essential for DNA replication.
- This complex comprises four subunits: p180, p68, p58, and p48.
- It possesses both DNA primase and DNA polymerase activities.
Purpose of the Study:
- To clone and characterize the subunits of the human and mouse DNA-polymerase-alpha--primase complex.
- To investigate the biochemical properties of individual subunits and their complexes.
- To determine the enzymatic activities of recombinant DNA-polymerase-alpha--primase complexes.
Main Methods:
- Cloning of cDNAs encoding DNA-polymerase-alpha--primase subunits from human and mouse.
- Over-expression of single polypeptides and co-expression of subunit complexes using baculovirus vectors.
- Purification of proteins and biochemical characterization of their enzymatic activities.
Main Results:
- High amino acid conservation was observed among mammalian proteins.
- The mouse p48 subunit (Mp48) alone exhibited primase activity.
- Co-expression of Mp48 and Mp58 subunits yielded a stable, highly active DNA primase.
- Co-expression of all four subunits produced substantial amounts of tetrameric DNA-polymerase-alpha--primase.
- Trimeric and tetrameric complexes demonstrated both primase and polymerase activities.
- Tetrameric recombinant DNA-polymerase-alpha--primase efficiently synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro.
Conclusions:
- The p48 subunit is crucial for primase activity.
- The DNA-polymerase-alpha--primase complex can be reconstituted from its subunits.
- Recombinant DNA-polymerase-alpha--primase exhibits robust enzymatic functions essential for DNA replication.