Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M

As transcription and replication use DNA as substrate, conflicts between transcription and replication can occur, leading to genome instability with direct consequences for human health. To determine how the two processes are coordinated throughout S phase, we characterize both processes together at high resolution. We find that transcription occurs during DNA replication, with transcription start sites (TSSs) not fully replicated along with surrounding regions and remaining under-replicated until late in the cell cycle. TSSs undergo completion of DNA replication specifically when cells enter mitosis, when RNA polymerase II is removed. Intriguingly, G2/M DNA synthesis occurs at high frequency in unperturbed cell culture, but it is not associated with increased DNA damage and is fundamentally separated from mitotic DNA synthesis. TSSs duplicated in G2/M are characterized by a series of specific features, including high levels of antisense transcription, making them difficult to duplicate during S phase.