DNA HELICASE ASSOCIATED WITH DNA-POLYMERASE ALPHA - ISOLATION BY A MODIFIED IMMUNOAFFINITY CHROMATOGRAPHY

We have developed a novel immunoaffinity method for isolating a DNA po lymerase-alpha-associated DNA helicase from the yeast Saccharomyces ce revisiae. Earlier we have reported the characterization of a DNA helic ase activity associated with the multiprotein DNA polymerase alpha com plex from yeast [Biswas, E. E., Ewing, C. M., & Biswas, S. B. (1993) B iochemistry 32, 3030-3027]. We report here the isolation of the DNA he licase from the DNA polymerase alpha (pol alpha) complex bound to an a nti-pol alpha immunoaffinity matrix. The DNA helicase activity eluted at approximately 0.35 M NaCl concentration. The eluted ATPase/helicase peak was further purified by size-exclusion high-performance liquid c hromatography (HPLC). At low ionic strength (50 mM NaCl), it remained associated with other proteins and eluted as a large polypeptide compl ex. At high ionic strength (500 mM NaCl), the helicase dissociated, an d the eluted ATPase/helicase fraction contained 90-, 60-, and 50-kDa p olypeptides. Photoaffinity cross-linking of helicase with ATP during t he isolation process demonstrated a 90-kDa polypeptide to be the likel y ATP binding component of the helicase protein. The DNA helicase has single-stranded DNA (ssDNA)-stimulated ATPase and dATPase activities. The ATPase activity was stimulated by yeast replication protein A (RPA ). The DNA helicase activity was stimulated by Escherichia coli ssDNA binding protein and RPA. The DNA helicase migrated on a DNA template i n the 5'-->3' direction which is also the overall direction of migrati on of pol alpha on the lagging strand of the replication fork. The pro cedure described here may prove equally useful in the isolation other DNA polymerase alpha-associated replication proteins, in addition to D NA helicase, from yeast and other eukaryotic cells.