Conflict resolution between the replisome and Pol II

The replisome and RNA polymerase II (Pol II) use the sample 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 several recently developed techniques, we determined in vivo the replication fork speed, direction, and interruption when the replisome met Pol II. Based on nsRNA-seq of the mouse embryonic fibroblasts (MEFs), we revealed that 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 20% reduction of II density in the S-phase-enriched MEFs compared to asynchronous MEFs was observed. These two means represent the means of temporal segregation from replication. A moderate and significant bias (about 17% difference) was found favoring the co-directional forks over head-on forks. The encounter between the replisome and Pol II was quite eventful. About 21% of the forks were aborted in the Pol II-dense TSS region. Although the majority of the forks could still pass the TSS region, they hesitated and slowed down when the tips of the forks approached the Pol II dense region. Paradoxically, the forks traveled faster in genome regions dotted with more Pol IIs as long as their tips were not near the latter, probably due to a higher degree of chromatin negative supercoiling near Pol IIs. Our study demonstrated that the mammalian cells relied on multiple measures, such as temporal segregation, co-direction preference, and fork abortion, to mitigate the danger of replication-transcription conflict, but all these measures operated only as auxiliaries. The majority of the forks could pass Pol IIs unscathed, indicating the existence of a so-far unidentified, very efficient conflict resolution mechanism.