Purification and characterization of DNA polymerase alpha-associated replication protein A-dependent yeast DNA helicase A

Biochemistry. 1997 Oct 28;36(43):13270-6. doi: 10.1021/bi9712910.


A novel, eukaryotic, hexameric DNA helicase that was earlier identified as a component of the multiprotein polymerase alpha complex [Biswas et al. (1993) Biochemistry 32, 13393-13398] has been purified to homogeneity and characterized. Thus far, our studies demonstrated that helicase A shares certain unique features of two other hexameric DNA helicases: the DnaB helicase of Escherichia coli and the T-antigen helicase of the SV40 virus. The helicase activity was stimulated by yeast replication protein A (RPA) and to a lower extent by E. coli single-stranded DNA binding protein (SSB). The helicase had an apparent molecular mass of 90 kDa, as determined by its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A tryptic peptide fragment of the polypeptide was sequenced followed by a BLAST search of GenBank with the tryptic peptide sequence. The search identified a 1.8 kb open reading frame previously designated as ykl017c on chromosome XI, that codes for a 78.3 kDa (683 amino acid) polypeptide. The important features of the polypeptide sequence of helicase A included a type I ATP/GTP binding motif, and a K E E R R L N V A M T R P R R sequence at the C-terminus that may be indicative of a nuclear localization signal which is required of a nuclear DNA helicase. The polypeptide sequence of helicase A appears to have homology to the DnaB helicase of E. coli (approximately 25%). The facts that these two helicases are vastly separated by evolution and retained similar structural and functional features, as demonstrated here, point to a possible significance of this limited homology. Although the amount of purified helicase A was limited, we have carried out necessary enzymatic characterization so that these data could be correlated with that of immunoaffinity-purified helicase A and recombinant helicase A expressed in heterologous systems.

Publication types

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

MeSH terms

  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / isolation & purification*
  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • DNA Helicases*
  • DNA Polymerase I / metabolism*
  • DNA Replication*
  • DNA-Binding Proteins / physiology*
  • DnaB Helicases
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Genes, Fungal
  • Molecular Sequence Data
  • Nucleotidases / metabolism
  • Photochemistry
  • Replication Protein A
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins


  • DNA-Binding Proteins
  • Fungal Proteins
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphate
  • DNA Polymerase I
  • Nucleotidases
  • Adenosine Triphosphatases
  • HCS1 protein, S cerevisiae
  • dnaB protein, E coli
  • DNA Helicases
  • DnaB Helicases

Associated data

  • SWISSPROT/P34243