Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA

Abstract

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.