PURIFICATION OF 2 DNA-DEPENDENT ADENOSINE-TRIPHOSPHATASES HAVING DNA HELICASE ACTIVITY FROM HELA-CELLS AND COMPARISON OF THE PROPERTIES OF THE 2 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 po sition of Q1 and exhibited a broader peak with higher activity than no rmal Q2 at the peak position of Q2 [Yanagisawa, J., Seki, M., Ui, M., and Enomoto, T. (1992) J. Biol. Chem. 267, 3585-3588]. We have purifie d two DNA-dependent ATPases Q1 and Q2 from HeLa cells and characterize d their properties in order to obtain a means to discriminate ATPase Q 1 from Q2 in XP-C cells. The apparent molecular masses of Q1 and Q2 on SDS-polyacrylamide gel electrophoresis were 73 and 100 kDa, respectiv ely. The two enzymes required a divalent cation for activity. DNA-depe ndent ATPase Q1 hydrolyzed ATP and dATP and Q2 hydrolyzed ATP preferen tially among the nucleotides tested. Both enzymes preferred single-str anded DNA as a cofactor. The DNA-dependent ATPase activity of Q2 was i nhibited 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 t hat of Q2. The DNA helicase activity of Q2 was about 150-fold higher t han that of Q1, when compared with units of ATPase activity. The direc tion of unwinding for Q1 was from 3' to 5' and that for Q2 was 5' to 3 ' with respect to the DNA to which the enzymes bound. Examinations bas ed on the differences in the properties of the two enzymes have indica ted that DNA-dependent ATPase Q1 is altered in XP-C cells, resulting i n the overlapping elution of Q1 and Q2.