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The Journal of Biochemistry
Vol. 115 (1994) No. 3 P 523-531


DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks designated Q 1 to Q 5 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 Qi and exhibited a broader peak with higher activity than normal Q 2 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 Q 1 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 200mM NaCl, whereas that of Q 1 was not affected by NaCl at concentrations up to 200mM. 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. The direction of unwinding for Q 1 was from 3' to 5' and that for Q2 was 5' to 3' with respect to the DNA to which the enzymes bound. Examinations based on the differences in the properties of the two enzymes have indicated that DNA-dependent ATPase Q1 is altered in XP-C cells, resulting in the overlapping elution of Q 1 and Q 2.

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