The dNTP synthesis inhibitor hydroxyurea (HU) can induce replication stress to slow replication elongation and thus greatly improve the resolution of EdU-marked DNA replication initiation (EdU-seq-HU) experiments. Recently, high-resolution Repli-seq was employed to profile initiation zones by dissecting the S phase into 16 fractions. Repli-seq has been applied in studies exploring the determination of replication timing, which have suggested that replication domains range from 400 to 800 kb in size. Chromatin immunoprecipitation-sequencing (ChIP-seq) of pre-replication complex (pre-RC) components is difficult because high-quality antibodies against the origin recognition complex (ORC) and mini-chromosome maintenance complex (MCM) are not available. Many attempts have been made to identify replication origins and initiation zones in mammalian cells. Replication origins have been well characterized in bacteria and yeast, but their locations in mammalian cells remain unclear. ĭNA replication occurs throughout the entire genome, in contrast to transcription, and tracing early DNA replication requires precise identification of genomic DNA replication origins and initiation zones. Previous studies suggest that transcription might affect DNA replication initiation, but the mechanism underlying this interaction in mammalian cells remains elusive. Therefore, early DNA replication and gene transcription both occur within active chromatin compartments, raising the pivotal question of how these processes are spatially and temporally coordinated to avoid replication-transcription collisions and subsequent DNA damage. Early DNA replication is initiated within active chromatin compartments, followed by elongation into inactive compartments. The mammalian genome is categorized into active and inactive compartments linked to key cellular processes according to chromatin activity and histone modifications, and transcription mainly occurs in active chromatin compartments. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability. RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions.
Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.
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