G-Quadruplex DNA suppresses Transcription during DNA Replication [CUT&Tag]

G-quadruplex DNA (G4-DNA) structures play crucial but incompletely understood roles in coordinating DNA replication and transcription. Here, we investigate the dynamics of G4-DNA during DNA replication and its functional interplay with transcription machinery in synchronized tumor cells. Using a double-thymidine block to arrest cells in early S phase, we profile G4-DNA structures and associated transcription factors through CUT&Tag, while monitoring transcriptional changes via PRO-seq. To mechanistically dissect these relationships, we perturb the system by overexpressing G4-DNA helicases (DHX36 and BRIP1), treating with the G4-destabilizing agent PhpC, and knocking down the novel G4-binding protein RFC3. Our integrated analysis reveals how G4-DNA spatially and temporally regulates replication-transcription coupling, with important implications for understanding genome stability in cancer. These findings provide new insights into the role of G4-DNA as a regulatory nexus between DNA replication and gene expression.