Three new DNA helicases from Saccharomyces cerevisiae.
At least six DNA helicases have been identified during fractionation of extracts from the yeast Saccharomyces cerevisiae. Three of those, DNA helicases B, C, and D, have been further purified and characterized. DNA helicases B and C co-purified with DNA polymerase delta through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography. DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl. All three helicases are DNA dependent ATPases with Km values for ATP of 190 microM, 325 microM, and 60 microM for DNA helicases B, C, and D, respectively. Their DNA helicase activities are comparable. They are 5'-3' helicases and have pH optima of 6.5-7 and Mg2+ optima of 1-2 mM. However, they differ in the nucleotide requirement for helicase action. Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP. On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor. These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.
Li
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Department of Biochemistry and Molecular Biophysics, Washington University, School of Medicine, St. Louis, MO 63110.
Yoder
B L
BL
Burgers
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PM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004258:DNA Polymerase III; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000502:physiology; D005656:Fungal Proteins / Q000302:isolation & purification; D018398:Homeodomain Proteins; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D051818:Replication Protein C