Initiator protein dimerization plays a key role in replication control of Vibrio cholerae chromosome 2

Abstract

RctB, the initiator of replication of Vibrio cholerae chromosome 2 (chr2), binds to the origin of replication to specific 12-mer sites both as a monomer and a dimer. Binding to 12-mers is essential for initiation. The monomers also bind to a second kind of site, 39-mers, which inhibits initiation. Mutations in rctB that reduce dimer binding increase monomer binding to 12-mers but decrease monomer binding to 39-mers. The mechanism of this paradoxical binding behavior has been unclear. Using deletion and alanine substitution mutants of RctB, we have now localized to a 71 amino acid region residues important for binding to the two kinds of DNA sites and for RctB dimerization. We find that the dimerization domain overlaps with both the DNA binding domains, explaining how changes in the dimerization domain can alter both kinds of DNA binding. Moreover, dimerization-defective mutants could be initiation-defective without apparent DNA binding defect. These results suggest that dimerization might be important for initiation beyond its role in controlling DNA binding. The finding that determinants of crucial initiator functions reside in a small region makes the region an attractive target for anti-V. cholerae drugs.

Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome

“…Binding was assessed for WT RctB (659 amino acids) and an N-terminal fragment consisting of amino acids 1 to 500 (RctB ΔC159). Previous work revealed that RctB ΔC159 binds to a 12-mer and a 39-mer with different affinities than WT ( 14 , 21 , 23 ), but it is not known if this RctB fragment binds to an 11-mer in a different manner than WT; this information might help explain why RctB ΔC159 is not subject to Inc-mediated negative regulation ( 14 , 21 , 23 ).…”

“…The mutant’s lack of inhibition by inc appears to reflect the higher affinity of ΔC159 mutant RctB than wild-type (WT) RctB for a 12-mer and its lack of binding to the 39-mer motifs found in oriCII ( 14 , 21 ). In addition, truncated forms of RctB have a lower capacity to dimerize than the full-length protein, and Chattoraj and colleagues have proposed that monomeric and dimeric forms of RctB promote and inhibit initiation at oriCII , respectively ( 23 ). The effects of the truncations are incompletely understood; nonetheless, their behavior has provided some insight into how RctB-mediated initiation at oriCII may be regulated ( 14 , 21 – 23 ).…”

Replication Initiation in Bacteria

“…Initiation from oriC2 requires RctB, which recognizes 11mer and 12mer iterons, and also a 39mer and a truncated form of the 39mer named the 29mer. The 39mer in the inc2 region and the 29mer bound by the monomeric form of RctB together with the DnaA box bound by V. cholerae DnaA regulate the frequency of initiation from oriC2 [37]. Adapted from Refs.…”

“…1). oriC2 contains an array of tandem repeats named the 11mer and 12mer that are bound by monomers and dimers of RctB [37,38]. Another DNA motif named the 39mer is specifically bound by monomeric RctB.…”

Management of multipartite genomes: the Vibrio cholerae model

“…RctB binds and hydrolyzes ATP but unlike DnaA, the ATP-bound form of RctB is inactive [14]. It binds to DNA as monomer or as a dimer [15,16]. Finally, RctB is most probably remodeled by chaperones DnaJ and DnaK similar to iteron-carrying plasmid's Rep initiators [15,17].…”