Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1’s ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway. Display omitted •BRCA1 is partitioned within TopBP1 condensates in a FANCJ-dependent manner•Chk1 is phosphorylated at the interfacial regions of TopBP1 biomolecular condensates•Optogenetic TopBP1 condensation system recapitulates this phenomenon•Light-activated Chk1 phosphorylation triggers cell cycle checkpoints without DNA damage Egger et al. implement their previously proposed ATR/Chk1 activation model by dissociating the functions of ATR and Chk1. Using reverse genetics and optogenetics, they show that Chk1 is phosphorylated at the interface of TopBP1 biomolecular condensates, triggering cell cycle checkpoints and slowing down replication fork progression.