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. Overall design: This study investigates the role of ??G4-DNA?? in regulating ??DNA replication??-coupled transcription by combining cell cycle synchronization, genetic perturbations, and multi-omics profiling in ??HCCLM3 hepatocellular carcinoma cells??. Using a ??double-thymidine block??, we synchronize cells to ??Early S phase?? to examine phase-specific dynamics of G4-DNA structures and their interplay with transcription machinery. To dissect mechanistic relationships, we (1) overexpress G4-DNA helicases (??DHX36/BRIP1??), (2) treat with the G4-destabilizing agent ??PhpC??, and (3) knockdown the novel G4-binding protein ??RFC3?? in synchronized cells. These perturbations are analyzed through ??CUT&Tag?? (profiling transcription-associated proteins like RNA Pol II-S2P/S5P and NELF complex) and ??PRO-seq?? (quantifying transcriptional output). Our integrated approach will delineate how G4-DNA modulates replication-transcription crosstalk, with implications for understanding genome stability in cancer.