| The Escherichia coli Rep protein has DNA-dependent ATPase activity and unwinds duplex DNA with an apparent 3{dollar}spprime{dollar} to 5{dollar}spprime{dollar} directionality. Limited treatment of unliganded and ssDNA-bound Rep protein (73 kDa) with trypsin results in cleavages in carboxyl (C)-terminal regions to give 68 kDa and 58 kDa polypeptides, respectively. The 68 kDa polypeptide retains the ability to bind ssDNA, hydrolyze ATP, and selectively unwind partial duplex DNA over RF DNA in the presence of bacteriophage nicking enzyme. These results suggest that the C-terminal end ({dollar}sim{dollar}5 kDa) of the Rep protein is not required for its helicase or ATPase activities, but is important for its interaction with bacteriophage nicking enzyme. The 58 kDa polypeptide displays a lower affinity for ssDNA, indicating that the 10 kDa region on the C-terminus facilitates the Rep protein binding to ssDNA. Both the 58 kDa and 68 kDa polypeptides are stabilized by the presence of nucleotides. Therefore, the ligand-dependent conformational changes of the Rep protein seem to be linked with its mechanism of unwinding.; We found that unliganded and nucleotide-bound Rep protein remains a monomer (73 kDa) up to concentrations of up to 8 {dollar}mu{dollar}M (monomer). The Rep protein forms a high molecular weight oligomer upon binding to d(pT){dollar}sb{lcub}rm n{rcub}{dollar} (n = 8-20) or a short duplex DNA. When the protein-DNA complex is crosslinked with dimethyl suberimidate, the crosslinked species is determined to be a dimer with an apparent molecular weight (M{dollar}sb{lcub}rm app{rcub}{dollar}) of 155 {dollar}pm{dollar} 5 kDa. Formation of the crosslinked Rep dimer when complexed with d(pT){dollar}sb{lcub}rm n{rcub}{dollar} (n = 8-20), and the decrease in M{dollar}sb{lcub}rm app{rcub}{dollar} of Rep-d(pT){dollar}sb{lcub}28{rcub}{dollar} as a function of d(pT){dollar}sb{lcub}28{rcub}{dollar} show that (1) DNA binding induces the dimerization of Rep protein, and (2) two Rep monomers do not bind contiguously on d(pT){dollar}sb{lcub}rm n{rcub}{dollar} for n {dollar}<{dollar} 20. The DMS-crosslinked Rep dimer retains the ability to hydrolyze ATP, unwind M13 RF DNA in the presence of f1 gene II protein, and bind ssDNA with increased affinity, indicating that the dimer is active as a helicase. A theoretical model is used to quantitatively describe the DNA-induced Rep dimerization. (Abstract shortened with permission of author.)... |
