DNA replication in vitro by recombinant DNA-polymerase-alpha-primase

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.