The results can be summarized as follows. SpoIIIE was
overexpressed and purified by virtue of an affinity tag. The purified protein
shows a DNA dependent ATPase activity. In tracking assays a positively
supercoiled product was generated, when ATP was replaced with a non-hydrolysable
analogue no product was generated. Taken together, these results suggest
that SpoIIIE can track along DNA in a reaction which requires the hydrolysis
of ATP. Further experiments confirm that the supercoiling is due to tracking
rather than alternative mechanisms (for example, wrapping of DNA). These
experiments demonstrate that SpoIIIE has the potential to pump DNA and,
taken together with genetic evidence, provide convincing evidence that
SpoIIIE is responsible for chromosome transfer during sporulation. Several
interesting questions remain unanswered, for example how is it that SpoIIIE
is able to ensure that the chromosome is transferred into the prespore and
not out of the prespore. |
