DNA helicase associated with DNA polymerase alpha: isolation by a modified immunoaffinity chromatography.
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)
Biswas
E E
EE
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201.
Chen
P H
PH
Biswas
S B
SB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D009994:Osmolar Concentration; D010777:Photochemistry; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012965:Sodium Chloride; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004257:DNA Polymerase II; D000251:Adenosine Triphosphatases; D004265:DNA Helicases