Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD

摘要: The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state active complex, its processivity mechanism unwinding. By tethering either or microscope coverslip surface, unwinding both linear natural circular plasmid by PcrA/RepD was followed real-time total internal reflection fluorescence microscopy. Visualization achieved fluorescent single-stranded DNA-binding protein. data show that PcrA, combination RepD, can unwind lengths single run, is as monomer. Although average rate similar assays, experiments revealed wide distribution speeds different molecules. several-fold slower than translocation on DNA, suggesting may proceed via partially passive mechanism. However, fastest dsDNA rates assays approached speed ssDNA.