The potential for transcription to impair DNA replication
Transcription does not stop during DNA replication and transcription-replication conflicts must arise since both processes coexist on chromosomes. Furthermore, the transcriptional machinery includes many DNA-bound protein complexes that might impair the movement of the replisomes even if active RNA polymerases are removed.
Whereas bacterial genes tend to be ordered co-directionally with DNA replication (Kunst et al., 1997; McLean et al., 1998; Rocha and Danchin, 2003), in S. cerevisiae almost half of the genes are transcribed opposite to the normal direction of DNA replication (referred to as ”head-on” genes), so encounters between the replisome and RNA polymerases must be frequent (Garcia-Muse and Aguilera, 2016; Goehring et al., 2023). Also unlike bacteria, there is little evidence that head-on orientated genes in yeast have higher mutation rates so the eukaryotic replisome must be adept at traversing transcriptional units (Sankar et al., 2016). In theory, DNA damage systems could alleviate transcription-replication collisions in head-on genes, but the DNA damage response is global and delays replication throughout the genome when forks stall, which must represent an emergency response rather than the default behaviour (reviewed in (McClure et al., 2022)). Similarly, while genome-wide profiling of DNA replication has provided evidence that the replisome pauses at highly expressed loci, the delays required for the replisome to wait until transcription completes at each head-on gene would be substantial (often >1 minute given average gene size and RNA pol II holoenzyme speed (Muniz et al., 2021)), whereas minimal pausing was detected (Azvolinsky et al., 2009; Claussin et al., 2022; Kara et al., 2021). Replication profiles show that in S. cerevisiae many replisomes travel 50 kb through gene rich regions in a normal S-phase lasting ~ 30 minutes (Kara et al., 2021), which is at the limit of travel given the measured yeast fork speed of 1.6-1.9 kb/min (Hodgson et al., 2007; Sekedat et al., 2010), so pauses must be short and infrequent. Therefore, the eukaryotic replisome must efficiently displace RNA polymerases, just as the bacterial replisome has been directly observed to do by electron microscopy (French, 1992).
Although we might imagine that head-on collisions between replication and transcription are inherently recombinogenic, extensive studies of the budding yeast ribosomal DNA show exactly the opposite. The unidirectional replication fork barrier (RFB) formed by Fob1 binds the ribosomal DNA downstream of the massively transcribed 35S region and prevents the replisome meeting oncoming RNA polymerase I head-on (Kobayashi and Horiuchi, 1996). However, replication forks stalled at the RFB are highly recombinogenic (Kobayashi et al., 1998; Stewart and Roeder, 1989), whereas in RFB-defective mutants replication forks proceed seemingly unimpeded head-on through the 35S region while recombination rate is vastly decreased and DNA damage marks are unchanged (Kara et al., 2021; Keszthelyi et al., 2023). This is also true for RNA polymerase II as reversion assays using a head-on or codirectional lys2 frameshift allele failed to demonstrate any significant difference in the overall rate of reversion between both orientations (Kim et al., 2007). There are exceptions however, since tRNA genes transcribed by RNA polymerase III do impede the replisome in head-on encounters, as detected by both 2D-gels and genome-wide methods and these encounters can be recombinogenic (Azvolinsky et al., 2009; Claussin et al., 2022; Deshpande and Newlon, 1996; Kara et al., 2021; Tran et al., 2017).
Regulatory proteins bound to DNA could also form obstacles for the replisome. The substantial machinery bound at promoters includes transcription factors and transcriptional activators (catalogued in (Rossi et al., 2021)), but as noted above genome-wide studies have revealed only the mildest impacts of these on replisome progression (Azvolinsky et al., 2009; Kara et al., 2021). Helicases including Rrm3 and Pif1 work assist the replisome in removing proteins, easing the path through difficult features including tRNAs (Azvolinsky et al., 2006; Azvolinsky et al., 2009; Claussin et al., 2022; Ivessa et al., 2003; Tran et al., 2017), and it has recently been found that replisome pausing at tRNA is primarily due to bound TFIIIC complex rather than transcriptionper se (Yeung and Smith, 2020). However, the mild replisome pausing at highly expressed RNA polymerase II genes is independent of Rrm3 (Azvolinsky et al., 2009), suggesting that the core replisome is able to remove protein obstacles associated with RNA polymerase II activity.
RNA polymerase II and associated factors therefore have the potential to impede DNA replication, and there is some evidence for this in extremely highly transcribed genes, but during rapid growth under nutrient rich conditions the replisome is very proficient in removing such obstacles, despite transcription being highest under such conditions. RNA polymerase III genes do have an effect on the replisome, but S. cerevisiae has dedicated helicases to ensure that replisome pausing at tRNA is minimal.