Over 10% of bacteria have expanded their genomes through the domestication of megaplasmids, which subsequently evolved into secondary chromosomes encoding core functions. A fundamental challenge of this genomic expansion is coordinating the replication of multiple replicons within a single cell cycle. In Vibrio cholerae, the replication of the secondary chromosome (Chr2) is intricately linked to the replication of the primary chromosome (Chr1) via a unique checkpoint sequence, crtS. This sequence binds to the initiator of Chr2, RctB. While crtS replication on Chr1 triggers Chr2 replication, the specific molecular dynamics remain elusive. To investigate this, we conducted a comprehensive genome-wide analysis of RctB binding patterns in V. cholerae across various cell cycle stages. Our findings show that RctB primarily binds to sites inhibiting replication initiation at the Chr2 origin (ori2). This inhibitory effect is counteracted when crtS replicates on Chr1, causing a shift in RctB binding to sites that activate replication at ori2. Structural analyses support the formation of diverse oligomeric states of RctB, coupled to the allosteric effect of DNA, which determine ori2 accessibility. We propose a synchronization model where, upon replication, crtS locally destabilizes the RctB inhibition complex, releasing the Chr2 replication origin.