Single-molecule DNA replication dynamics under WEE1 inhibition in wild-type and TRESLIN-stabilized HCT116 cells

We analyzed two engineered HCT116 lines expressing mClover-tagged TRESLIN from the endogenous locus: a wild-type TRESLIN allele and a stabilized ΔSBI TRESLIN mutant. Cells were treated with the WEE1 inhibitor MK-1775 (1 µM) or vehicle and sequentially pulse-labeled with EdU and BrdU, followed by a thymidine chase, to generate replication tracts for long-read nanopore sequencing.

High–molecular weight genomic DNA was prepared and sequenced on an Oxford Nanopore PromethION platform, and reads were aligned to the human reference genome (hg38/GRCh38). Replication features (leftward/rightward forks, initiation events, and termination events) and tract lengths were inferred from EdU/BrdU segmentation using DNAscent forkSense, enabling quantification of fork directionality and fork rates across conditions.