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

Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII.