DNA helicase associated with DNA polymerase alpha: isolation by a modified immunoaffinity chromatography

Biochemistry. 1993 Dec 14;32(49):13393-8. doi: 10.1021/bi00212a003.

Abstract

We have developed a novel immunoaffinity method for isolating a DNA polymerase alpha-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 alpha 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 approximately 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'-->3' direction which is also the overall direction of migration of pol alpha on the lagging strand of the replication fork.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • Binding Sites
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Cross-Linking Reagents
  • DNA Helicases / isolation & purification*
  • DNA Helicases / metabolism
  • DNA Polymerase II / metabolism*
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / pharmacology
  • Escherichia coli / chemistry
  • Osmolar Concentration
  • Photochemistry
  • Replication Protein A
  • Saccharomyces cerevisiae / enzymology*
  • Sodium Chloride
  • Templates, Genetic

Substances

  • Cross-Linking Reagents
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Replication Protein A
  • Sodium Chloride
  • Adenosine Triphosphate
  • DNA Polymerase II
  • Adenosine Triphosphatases
  • DNA Helicases