Purification and characterization of DNA polymerase alpha-associated replication protein A-dependent yeast DNA helicase A.
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.
Biswas
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SB
Department of Molecular Biology, Science Center, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA. biswassb@umdnj.edu
Chen
P H
PH
Biswas
E E
EE
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004265:DNA Helicases; D004256:DNA Polymerase I / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000502:physiology*; D053488:DnaB Helicases; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009708:Nucleotidases / Q000378:metabolism; D010777:Photochemistry; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004256:DNA Polymerase I; D009708:Nucleotidases; D000251:Adenosine Triphosphatases; C506734:HCS1 protein, S cerevisiae; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases