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, regulates expression of its own gene and another partition gene, parB, from a promoter upstream of parA. The ATP-dependent ParA DNA binding activity to the par promoter is thought to mediate this regulation. An alternate purification for ParA is presented. This highly purified ParA was used to examine 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. Non-hydrolyzable ATP analogues as well as ADP stimulated binding more than ATP, suggesting that the act of hydrolysis is coupled to release of the DNA. Glycerol gradient sedimentation and chemical cross-linking experiments suggest that ParA exists in an monomer-dimer equilibrium that is shifted toward dimer formation by adding 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 effects on oligomerization.
Davey
M J
MJ
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Funnell
B E
BE
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004274:DNA, Recombinant / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C052883:chromosome partition proteins, bacterial; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate