Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif

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Abstract
The initiator protein of the plasmid pPS10, RepA, has a putative hellx-turn-hellx (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. 7lsqb;D. Garcia de viedma, etal. (1996) EMBOJ. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations In RepA, associated with deficiencies in autoregulation, map either at the putative HTH motif or In its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and ortV regions in vitro. Revertats of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be Involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same.