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

Abstract

We have established a novel procedure to purify calf thymus DNA polymerase δ from cytoplasmic extracts. The enzyme has typical properties of a DNA polymerase δ 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 δ , DNA polymerase α-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase δ separated from the coeluting DNA polymerase α and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase δ was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase δ was resistant to the DNA polymerase α 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 δ and DNA polymerase α might act coordinately at the replication fork as leading and lagging strand replicases, respectively.