Transcription shapes DNA replication initiation to preserve genome integrity

We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones (ERIZs) in both mouse and human cells. The identified ERIZs fall in open chromatin compartments while are mutually exclusive with transcription elongation and occupy mainly non-transcribed regions adjacent to transcribed regions. Furthermore, we reveal that RNA polymerase II actively redistributes the chromatin-encircled mini-chromosome maintenance (MCM) complex but not the origin-recognition complex (ORC) to actively restrict early DNA replication initiation outside of transcribed regions. The coupling of RNA polymerase II and MCM is further validated by detected MCM accumulation and DNA replication initiation after RNA polymerase II stalling via anchoring nuclease-dead Cas9 at the transcribed genes. Importantly, we also find that the orchestration of DNA replication initiation by transcription can efficiently prevent gross DNA damage. We have finished the NAIL-seq with EdU and BrdU dual labeling replicating DNA with K562/GM12878 cell line, NAIL-seq with EdU label only under HU treatment(EdU/HU) of K562/GM12878/mESC/mSB cells. We have finished NAIL-seq EdU/HU in K562 cells under transcription inhibition via a-amanitin/DRB, NAIL-seq EdU/HU in mouse spleenic B cells under a-amanitin, NAIL-seq EdU/HU in mESC RNA Pol2 degron system. We have finished ORC2 ChIP-seq and MCM5 ChIP-seq in both untreated cells and cells under transcription inhibition.