THE P1 PLASMID PARTITION PROTEIN PARA - A ROLE FOR ATP IN SITE-SPECIFIC DNA-BINDING

ParA, a P1 protein required for partition of the prophage plasmid, reg ulates expression of its own gene and another partition gene, parB, fr om a promoter upstream of parA. The ATP-dependent ParA DNA binding act ivity to the par promoter is thought to mediate this regulation. An al ternate purification for ParA is presented. This highly purified ParA was used to enamine ParA DNA binding activity using DNase I protection assays. At high concentration, ParA bound to the par promoter in the absence of ATP, demonstrating that although ATP stimulates, it is not required for DNA binding. Nonhydrolyzable ATP analogues as well as ADP stimulated binding more than ATP, suggesting that the act of hydrolys is is coupled to release of the DNA. Glycerol gradient sedimentation a nd chemical cross-linking experiments suggest that ParA exists in an m onomer-dimer equilibrium that is shifted toward dimer formation by add ing ATP or ADP. These observations lead to the proposal that the more active DNA binding form of ParA is a dimer and that the effects of ATP and ParA concentration on DNA binding are a direct result of their ef fects on oligomerization.