Purification of two DNA-dependent adenosinetriphosphatases having DNA helicase activity from HeLa cells and comparison of the properties of the two enzymes.
DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks designated Q1 to Q5 by FPLC Mono Q column chromatography. In our previous study, we observed that crude extracts prepared from xeroderma pigmentosum complementation group C (XP-C) cells contained no DNA-dependent ATPase activity at the peak position of Q1 and exhibited a broader peak with higher activity than normal Q2 at the peak position of Q2 [Yanagisawa, J., Seki, M., Ui, M., & Enomoto, T. (1992) J. Biol. Chem. 267, 3585-3588]. We have purified two DNA-dependent ATPases Q1 and Q2 from HeLa cells and characterized their properties in order to obtain a means to discriminate ATPase Q1 from Q2 in XP-C cells. The apparent molecular masses of Q1 and Q2 on SDS-polyacrylamide gel electrophoresis were 73 and 100 kDa, respectively. The two enzymes required a divalent cation for activity. DNA-dependent ATPase Q1 hydrolyzed ATP and dATP and Q2 hydrolyzed ATP preferentially among the nucleotides tested. Both enzymes preferred single-stranded DNA as a cofactor. The DNA-dependent ATPase activity of Q2 was inhibited by 90% in the presence of 200 mM NaCl, whereas that of Q1 was not affected by NaCl at concentrations up to 200 mM. Both enzymes had DNA helicase activity, that of Q1 being more resistant to NaCl than that of Q2. The DNA helicase activity of Q2 was about 150-fold higher than that of Q1, when compared with units of ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Seki
M
M
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
Yanagisawa
J
J
Kohda
T
T
Sonoyama
T
T
Ui
M
M
Enomoto
T
T
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data
D000251:Adenosine Triphosphatases; D004265:DNA Helicases