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

Abstract

Summary 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.

Protocol for analysis of G2/M DNA synthesis in human cells

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Figure 1 FACS analysis of S-phase progression following cells synchronization by serum starvation The top panel showing the FACS results using propidium iodide (PI) and BrdU after serum starvation (0 h) (bottom left, G0/G1 phase; top, S phase; bottom right, G2 phase); the bottom panel showing the quantification of cells in each stage; n = 3; average mean +/− standard error of the mean. (Adapted from Wang et al. 2021 ).

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“…Cells that are both in G2 by DNA content and pS10-H3 positive (highlighted in the box) are sorted followed by BrdU sequencing. (Adapted from Wang et al. 2021 ).…”

“…This method can also be used to study the role of DNA replication or transcription-associated factors in affecting G-MiDS levels in different cell lines. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) .…”

Consequences and Resolution of Transcription–Replication Conflicts

“…Interestingly, the relative locations of these elements along eukaryotic chromosomes are not random, but efficiently firing replication origins tend to overlap with transcription start sites (TSSs) of highly transcribed genes, thereby promoting a global CD bias of transcription and replication [ 10 , 11 ]. Although this strategy can effectively minimize HO-TRCs, considered to be the more harmful and deleterious type of TRC in bacteria, yeast and higher eukaryotes [ 12 , 13 , 14 , 15 ], a recent study showed that a subset of TSSs remains under-replicated and relies on G2/M DNA synthesis (G-MiDS) to complete replication and prevent DNA damage in mitosis [ 16 ]. These G-MiDS hotspots remain persistently under-replicated in the S-phase due to RNAP complexes at the TSS that block replication fork progression.…”