Open AccessDNA replication in vitro by recombinant DNA‐polymerase‐α‐primase
Author(s) -
STADLBAUER Franz,
BRUECKNER Andrea,
REHFUESS Christoph,
ECKERSKORN Christoph,
LOTTSPEICH Friedrich,
FÖRSTER Verena,
TSENG Ben Y.,
NASHEUER HeinzPeter
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18925.x
Subject(s) - primase , dna polymerase , dna clamp , dna polymerase ii , biology , microbiology and biotechnology , polymerase , dna replication , dna polymerase i , recombinant dna , dna , primer (cosmetics) , biochemistry , chemistry , gene , polymerase chain reaction , reverse transcriptase , organic chemistry
DNA‐polymerase‐α‐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‐α‐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‐α‐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‐α‐primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA‐polymerase‐α‐primase synthesized double‐stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.