PURIFICATION AND CHARACTERIZATION OF PRIMER RECOGNITION PROTEINS FROM HELA-CELLS

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase α complex in HeLa cells. Purified PRP is free of DNA polymerases α, ß, and δ, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36000 Da (PRP 1) and 41 000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 Å and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase a on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase α, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids. © 1990, American Chemical Society. All rights reserved.