Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase alpha complex.
We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Biswas
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EE
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201.
Ewing
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CM
Biswas
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SB
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases / Q000378:metabolism*; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010777:Photochemistry; D011067:Poly dA-dT / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013698:Templates, Genetic
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011067:Poly dA-dT; D008345:Manganese; D000255:Adenosine Triphosphate; D004257:DNA Polymerase II; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium