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

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 is 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 .alpha. inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase .alpha. 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.