DNA-REPLICATION IN-VITRO BY RECOMBINANT DNA-POLYMERASE-ALPHA-PRIMASE

Citation
F. Stadlbauer et al., DNA-REPLICATION IN-VITRO BY RECOMBINANT DNA-POLYMERASE-ALPHA-PRIMASE, European journal of biochemistry, 222(3), 1994, pp. 781-793
Citations number
56
Categorie Soggetti
Biology
ISSN journal
00142956
Volume
222
Issue
3
Year of publication
1994
Pages
781 - 793
Database
ISI
SICI code
0014-2956(1994)222:3<781:DIBRD>2.0.ZU;2-9
Abstract
DNA-polymerase-alpha-primase complex contains four subunits, p180, p68 , p58, and p48, and comprises a minimum of two enzymic functions. We h ave cloned cDNAs encoding subunits of DNA-polymerase-alpha-primase fro m human and mouse. Sequence comparisons showed high amino acid conserv ation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovi rus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase ac tivity. Purification of co-expressed Mp48 and Mp58 subunits yielded st able DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-polymerase-alpha-p rimase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric a nd trimeric DNA-polymerase-alpha-primase complexes showed both DNA pri mase and DNA polymerase activities. The tetrameric recombinant DNA-pol ymerase-alpha-primase synthesized double-stranded M13 DNA and replicat ed polyoma viral DNA in vitro efficiently.