DNA replication encounters numerous chromatin metabolic activities during the elongation process, yet the organization of multiple replication forks in mammalian cells and the impact of chromatin structures on DNA replication remain poorly understood. To address this, we developed the replication-associated in situ HiC (Repli-HiC) method to capture chromatin interactions involving nascent DNA, identifying over 2,600 fountain-like chromatin contact structures, indicative of replication fork coupling in human cells. Here, our findings demonstrate that two replication forks can be tethered in various ways, including architectural tethering by Cohesin and functional tethering by AND1. Dysfunction in Cohesin results in the failure to couple adjacent replication forks, while increasing the coupling of distal replication forks. Moreover, perturbation of fork coupling leads to fork instability, failure to restart stalled forks, and an increase in DNA breaks and chromosomal translocations, particularly enriched in chromatin fountain regions. These findings reveal the molecular mechanisms and physiological roles of fork coupling, offering new insights into the role of 3D genomics in DNA replication progression within mammalian cells.

replication forks, fork coupling, genome stablity