Characterization of Escherichia coli Rep protein-DNA interactions

Abstract

The Escherichia coli Rep protein has DNA-dependent ATPase activity and unwinds duplex DNA with an apparent 3' to 5' directionality. Biochemical and biophysical aspects of the Rep helicase-DNA interactions were examined in order to determine the mechanism of unwinding by Rep helicase. 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, respectibely. 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 (~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 low affinity of 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. The effect of DNA biding on the assembly state of the Rep protein is also investigated, and we find that unliganded or nucleotide-bound Rep protein remains a monomer (73kDa) up to concentration of up to 8 μM (monomer). The Rep protein forms a high molecular weight oligomer upon binding to d(pT)n (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[app]) of 155[plus or minus]5 kDa. Formation of the crosslinked Rep dimer when complexed with d(pT)n (n=8-20), and the decrease in M[app] of Rep-d(pT028 as a function of d(pT)28 show that (1) DNA binding induces the dimerization of Rep protein, and (2) two Rep monomers do not bind contiguously on d(pT)n for n<20...