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Nucleic Acids Research, 1988, Vol. 16, No. 14 6279-6295
© 1988

Articles

Calf thymus DNA polymerase {delta}: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase {alpha}, a DNA dependent ATPase and proliferating cell nuclear antigen

Federico Focher, Silvio Spadari1, Barbara Ginelli, Michael Hottiger, Max Gassmann and Ulrich Hübscher*

Department of Pharmacology and Biochemistry, University of Zürich-Irchel Winterthurerstrasse 190, CH-8057 Zürich, Switzerland 1Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207, I-27100 Pavia, Italy

*To whom correspondence should be addressed

Received February 22, 1988. We have established a novel procedure to purify calf thymus DNA polymerase {delta} from cytoplasmic extracts. The enzyme has typical properties of a DNA polymerase {delta} including a 3'->>5' exonuclease activity and efficiently replicates natural occuring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase {delta} , DNA polymerase {alpha}-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase {delta} separated from the coeluting DNA polymerase {alpha} and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase {delta} was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase {delta} was resistant to the DNA polymerase {alpha} inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase a monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase {delta} and DNA polymerase {alpha} might act coordinately at the replication fork as leading and lagging strand replicases, respectively.


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 J. Biol. Chem.Home page
R. Mossi, E. Ferrari, and U. Hubscher
DNA Ligase I Selectively Affects DNA Synthesis by DNA Polymerases delta  and epsilon  Suggesting Differential Functions in DNA Replication and Repair
J. Biol. Chem., June 5, 1998; 273(23): 14322 - 14330.
[Abstract] [Full Text] [PDF]


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