Dissociation and Reconstitution of a DNA Polymerase α-Primase Complex

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Abstract
The conditions for dissociation of the DNA polymerase α-primase complex (DNA polymerase α1) have been examined. It was revealed that 50% ethylene glycol effectively dissociated the complex. The dissociated DNA polymerase and primase were purified to eliminate cross-contaminating activities by column chromatography using buffers containing 50% ethylene glycol. The sedimentation coefficients of the purified DNA polymerase and primase were 7.1S and 5.7S, respectively. These two enzymes were mixed in the presence of 20% ethylene glycol and the mixture was sedimented through a glycerol gradient containing no ethylene glycol. The DNA polymerase and primase activities co-sedimented at 9.1S which corresponds to the S value of intact α1, indicating the reconstitution of the DNA polymerase α-primase complex.