Mg2+, Asp-, and Glu--effects in the processive and distributive DNA relaxation catalyzed by a eukaryotic...

The salt requirement for the catalysis of DNA relaxation carried out by a eukaryotic DNA topoisomerase I from Candida was reexamined with plasmid pBR322 DNA. Two levels of analysis were considered: the initial velocity of the overall reaction and the mode of this reaction (processivity vs distributivity). When looking at the monovalent salts from the first level, the replacement of Cl- by Glu- or Asp- greatly enhanced the salt range over which the enzyme was active. Moreover, the initial velocity reached an optimal value for a higher salt concentration in this case. For the cationic counterpart, K+ was a little more effective than Na+ and much more so than NH4+. Addition of 4 mM magnesium chloride affected both the range and the optimum of the initial velocity differentially, depending upon the monovalent salt, but with a general stimulating tendency. On the other hand, when the Mg2+ salt was varied, substitution of chloride by aspartate enhanced the optimum of the initial velocity for a fixed KCl concentration. In addition, magnesium aspartate (MgAsp2) and magnesium glutamate (MgGlu2) allowed the reaction to occur even without monovalent salt and over an extended range. Magnesium was also shown to directly interact with the general catalysis (Kd = 2.5 mM). From the second level of analysis, the presence of Mg2+ (except with NH4Glu), the substitution of Cl- by Glu- or Asp-, and a lower monovalent salt concentration than that used for the velocity optimum were required to promote the processive mode.(ABSTRACT TRUNCATED AT 250 WORDS)