The P1 plasmid partition protein ParA, roles for ATP binding and hydrolysis in plasmid partition

grantor: University of TorontoThe P1 prophage plasmid is stably maintained in its bacterial host despite its low copy number. This stability is due in part to an active partition system, par, that encodes the genes for two proteins, ParA and ParB, as well as a cis acting site, parS. ParA is required for at least two roles during partition in vivo. First, ParA represses expression of its own gene as well as parB from a promoter upstream of parA. This repression by ParA is stimulated by ParB. There is a second requirement for ParA that is thought to reflect a direct, as yet undefined role for ParA in the partition process. In vitro, ParA is an ATPase that is stimulated by ParB and by non-specific DNA. In addition, ParA binds the par operon promoter region, consistent with ParA's repressor activity in vivo. In this thesis I examine the effects of ATP binding and hydrolysis on ParA, particularly on ParA interaction with parOP and with ParB. I show that adenine nucleoside di- and triphosphates stimulate ParA DNA binding. ATP hydrolysis is not required for ParA DNA binding and, in fact, inhibits ParA DNA binding. The effects of ATP binding and hydrolysis on ParA-parOP interaction are correlated with changes in ParA conformation including, but not limited to, ParA dimerization. I have also found that ParB stimulates ParA DNA binding in vitro, consistent with ParB's role as a co-repressor in vivo. ParB stimulation of ParA DNA binding requires ATP hydrolysis. However, preliminary results suggest that ATP hydrolysis is not required for physical association of ParA and ParB, therefore ATP must exert its effect on ParA-ParB interaction via a different mechanism. My results have led to a model of ParA repressor function in which ATP binding and ATP hydrolysis have separable roles and have led to the suggestion that ATP binding and hydrolysis have separable roles in ParA's partition function as well.Ph.D