Mammalian Strategies for Replication-Transcription Conflict Resolution

The replisome and RNA polymerase II (Pol II) use the same DNA template during the S phase. Inevitably, there is a conflict between these two machineries when they try to pass each other to accomplish their respective tasks. How such a conflict is resolved, especially in mammalian cells, is a quintessential biological question of which we only have a limited understanding. Using a novel DNAscent and several established sequencing techniques, we quantitatively assessed several conflict resolution strategies used in mouse embryonic fibroblasts (MEFs). A tiny fraction of all the genes, i.e., about 1.5%, downregulated their transcription during the S-phase compared to the G1 phase. A moderate reduction of Pol II density on the DNA template in the S-phase-enriched MEFs compared to asynchronous MEFs was observed. These two collectively represented a small degree of temporal segregation between the replication and transcription. DNAscent revealed a 17% bias in favor of the co-directional forks over head-on forks. About 25.6 % of the forks were aborted near the Pol II-dense transcription start sites (TSSs) region. The majority of the forks could still pass the TSS region. However, among those who passed, their speed was slower for those with tips closer to the TSS, suggesting that the passing was not uneventful. Paradoxically, there was a tendency for the forks to travel faster in genome regions dotted with more Pol IIs. Most of these regions had a higher degree of negative supercoiling (unwound). Employing a system with artificially increased Pol II density in the chromatin, we established a causal relationship between Pol II density and negative supercoiling/fork speed. Our study demonstrated that the mammalian cells relied on multiple measures, such as temporal segregation, co-direction preference, and fork abortion, to resolve the replication-transcription conflict, but all these measures operated only as auxiliaries. The majority of the forks could pass Pol IIs unscathed, albeit with some difficulties, indicating the existence of a so-far unidentified, very efficient conflict resolution mechanism.