DNA replication in vitro by recombinant DNA-polymerase-alpha-primase.
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.
Institut für Biochemie d. LMU München, Germany.
F
F
Stadlbauer
A
A
Brueckner
C
C
Rehfuess
C
C
Eckerskorn
F
F
Lottspeich
V
V
Förster
B Y
BY
Tseng
H P
HP
Nasheuer
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D019915:DNA Primase; D004261:DNA Replication; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011120:Polyomavirus / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014779:Virus Replication
D011994:Recombinant Proteins; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases