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 identifie d as a component of the multiprotein polymerase a complex [Biswas et a l. (1993) Biochemistry 32, 13393-13398] has been purified to homogenei ty 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 replicat ion protein A (RPA) and to a lower extent by E. coli single-stranded D NA binding protein (SSB), The helicase had an apparent molecular mass of 90 kDa, as determined by its mobility on sodium dodecyl sulfate-pol yacrylamide gel electrophoresis. A tryptic peptide fragment of the pol ypeptide 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 yk1017e on chromosome XI, that codes fo r a 78.3 kDa (683 amino acid) polypeptide. The important features of t he polypeptide sequence of helicase A included a type I ATP/GTP bindin g motif, and a K E E R R L N V A M T R P R R sequence at the C-terminu s that may be indicative of a nuclear localization signal which is req uired of a nuclear DNA helicase. The polypeptide sequence of helicase A appears to have homology to the DnaB helicase of E, coli (similar to 25%). The facts that these two helicases are vastly separated by evol ution and retained similar structural and functional features, as demo nstrated here, point to a possible significance of this limited homolo gy. Although the amount of purified helicase A was limited, we have ca rried out necessary enzymatic characterization so that these data coul d be correlated with that of immunoaffinity-purifed helicase A and rec ombinant helicase A expressed in heterologous systems.