DNA helicase associated with DNA polymerase alpha: isolation by a modified immunoaffinity chromatography
1993
Biswas, E.E. | Chen, P.H. | Biswas, S.B.
We have developed a novel immunoaffinity method for isolating a DNA polymerase-associated DNA helicase from the yeast Saccharomyces cerevisiae. Earlier we have reported the characterization of a DNA helicase activity associated with the multiprotein DNA polymerase a complex from yeast [Biswas, E. E., Ewing, C. M., & Biswas, S. B. (1993) Biochemistry 32, 3030-3027]. We report here the isolation of the DNA helicase from the DNA polymerase alpha (pol alpha) complex bound, to an anti-pol alpha immunoaffinity matrix. The DNA helicase activity eluted at -0.35 M NaCl concentration. The eluted ATPase/helicase peak was further purified by size-exclusion high-performance liquid chromatography (HPLC). At low ionic strength (50 mM NaCl), it remained associated with other proteins and eluted as a large polypeptide complex. At high ionic strength (500 mM NaCl), the helicase dissociated, and the eluted ATPase/helicase fraction contained 90-, 60-, and 50-kDa polypeptides. Photoaffinity cross-linking of helicase with ATP during the isolation process demonstrated a 90-kDa polypeptide to be the likely 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 in the 5' leads to 3' direction which is also the overall direction of migration of pol a on the lagging strand of the replication fork. The procedure described here may prove equally useful in the isolation other DNA polymerase alpha-associated replication proteins, in addition to DNA helicase, from yeast and other eukaryotic cells.
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