Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

Teresa Dı́Az-López,Marta Lages-Gonzalo,Ana Serrano-López,Carlos Alfonso,Germán Rivas,Ramón Dı́Az-Orejas,Rafael Giraldo

doi:10.1074/jbc.m212024200

Abstract

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.

Highlights

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10
The rationale behind the new Leu 3 Ala mutations is as follows. (i) They would impair a possible dimerization through the putative LZ, because the side chain of Ala is smaller and less hydrophobic than that of Leu. (ii) They would necessarily disrupt the hydrophobic network linking ␣1, through Leu-12 and Leu-19, to the rest of the WH1 domain, mainly Trp-94. (iii) Mutations would allow ␣1 to move freely, resulting in an extended conformation of the proposed jack-knife
RepA-2L2A: a Folding Intermediate in the Pathway from Repressor Dimers to Initiator Monomers—In this paper we have described the design of RepA-2L2A, a mutant in the protein that initiates DNA replication of the Pseudomonas plasmid pPS10, based on the available three-dimensional structure of the monomer of a homologous initiator [24]

Summary

Introduction

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. Radiolabeled DNA fragments, including either repA operator sequence (Fig. 3A) or the four iterons found at pPS10 origin of replication (Fig. 3B), were incubated with increasing amounts of pure RepA protein, either WT (His6-tagged or untagged) or mutant 2L2A.

Results

Conclusion