PROCESSIVITY OF URACIL DNA GLYCOSYLASE

The purpose of this study was to determine the mechanism by which uracil DNA glycosylase locates uracil residues within double-stranded DNA. Using reaction conditions that contained low salt concentrations, the addition of uracil DNA glycosylase to plasmid DNAs containing multiple, randomly incorporated uracils resulted in the accumulation of form III DNA while unreacted form I DNA was still present. These data suggested that the enzyme utilizes a one-dimensional DNA-scanning mechanism such that this linear DNA arose by the accumulation of many single-strand breaks within the plasmid prior to enzyme dissociation. Reactions containing higher concentrations of uracil DNA glycosylase revealed a further accumulation of form III DNA after all form I DNA had been lost. These results suggested a partial (1.5-2 kb) enzyme processivity since the enzyme does not incise at all uracil bases on the DNA molecule prior to dissociation from that DNA. Since DNA scanning is regulated by electrostatic interactions, the processivity of the enzyme was evaluated through kinetic analyses of incision at various salt concentrations. At NaCl concentrations (< 50 mM), a significant amount of form III DNA accumulated while there were still unreacted form I DNAs present. In contrast, the accumulation of form III DNA was delayed at higher salt concentrations and the overall accumulation of form III DNA was less than that monitored at lower salt concentrations. DNAs were also analyzed by denaturing agarose gel electrophoresis in order to measure the average distance between strand breaks. Southern hybridizations showed a greater accumulation of breaks in DNAs that were reacted with the uracil DNA glycosylase at the lower salt concentrations, confirming a partial processivity for the enzyme.