Single-Molecule Imaging Reveals the Mechanism of Bidirectional Replication Initiation in Metazoa

Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG (Cdc45•Mcm2-7•GINS) helicases at each origin. This requires several firing factors (including TopBP1, RecQL4, DONSON) whose exact roles remain unclear. How two helicases are correctly assembled and activated at every single origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered a surprisingly dynamic picture of initiation. Firing factors transiently bind origins but do not travel with replisomes. Two Cdc45 simultaneously arrive at each origin and two GINS are recruited together, even though neither protein can dimerize. The synchronized delivery of two GINS is mediated by DONSON, which acts as a dimerization scaffold. We show that RecQL4 promotes DONSON dissociation and facilitates helicase activation. The high fidelity of bidirectional origin firing can be explained by a Hopfield-style kinetic proofreading mechanism.