Transcription provides mutational vulnerabilities and opportunities for repair
Although transcription generates torsional stress, yeast RNA polymerases are less sensitive to this than we might expect: even cells lacking both topoisomerase I (Top1) and topoisomerase II (Top2) show defective RNA polymerase I transcription but surprisingly minor effects on RNA polymerase II and RNA polymerase III activity (Brill et al., 1987; Brill and Sternglanz, 1988). However, transcription generates significant supercoiling which is a Top1 substrate, and Top1 errors in the form of short deletions can form up to 50% of mutations in highly transcribed sequences (Lippert et al., 2011; Takahashi et al., 2011). These arise both through mis-processing of trapped Top1 cleavage complexes in tandem repeats and through Top1 cleaving at ribonucleotides (Cho et al., 2013). Ribonucleotides are mistakenly incorporated in the genome during replication but usually removed by RNase H2 (Huang et al., 2015; Williams and Kunkel, 2014; Williams et al., 2013), however processing of ribonucleotides by Top1 results in a non-ligatable end subject to potentially error prone repair (Huang et al., 2015; Kim et al., 2011). Top1-mediated mutations are therefore a significant danger to the integrity of coding sequences, though surprisingly these mutations are biased to the non-template strand (Cho et al., 2015), and Top1 overexpression in yeast has been associated with mutagenesis and increased DNA damage (Sloan et al., 2017).
The passage of RNA polymerase II along the template strand creates a bubble of single stranded DNA (ssDNA) that is also likely to increase mutation on the non-template strand as ssDNA is innately more prone to endogenous chemical damage. For instance, spontaneous depurination and depyrimidination occur four times more frequently in ssDNA than in dsDNA (Billen, 1990), cytosine deamination to uracil forms over 100-fold faster (Lindahl, 1993), and base alkylation occurs more frequently in ssDNA (Fu et al., 2012); indeed recent genome-wide studies have confirmed that the non-template strand is more frequently altered by alkylating agents and through spontaneous cytosine deamination (Mao et al., 2017; Williams et al., 2023).
In contrast, the genetic stability of the template strand is enhanced by transcription due to efficient machinery for repairing DNA lesions that are encountered by RNA polymerase II. Many lesions can block the progression of RNA polymerase including intra and inter-strand crosslinks, DNA-protein crosslinks, cyclopurines and abasic sites (Brooks et al., 2000; Fielden et al., 2018; Jung and Lippard, 2007; Tornaletti et al., 2006). When elongating RNA pol II encounters such impediments, the remodelling factor Rad26 binds to the polymerase and the DNA sequence upstream. The ATPase activity of Rad26 allows a forward translocation of RNA polymerase II, facilitating the bypassing of non-bulky lesions or benign obstacles (Duan et al., 2020; Xu et al., 2017), but when transcription-blocking lesions (TBLs) are encountered the holoenzyme can engage the transcription-coupled nucleotide excision repair (TC-NER) system. In yeast, TC-NER is activated by Rad26 or occasionally the non-essential Rpb9 RNA pol II subunit (reviewed in (Li, 2015)) , depending on expression level (Duan et al., 2020; Li and Smerdon, 2002, 2004). While the RNA polymerase traverses the block or, for severe lesions, is ubiquitinated and degraded, the cell initiates a repair process involving excision of nucleotides surrounding the lesion. Then, DNA polymerases including PolΓ and Polε are recruited to fill in the gap and the newly synthesised DNA is ligated before the transcription restarts (reviewed in (Gregersen and Svejstrup, 2018)).
Although yeast lacking TC-NER components show little phenotype under ideal conditions, the long term importance of this system in maintaining transcriptional homeostasis and suppressing mutations is underlined by the phenotypes of human diseases caused by TC-NER mutations including UV-sensitive syndrome, Xeroderma Pigmentosum and Cockayne syndrome, which is usually caused by mutations in the human orthologue of Rad26 (Lans et al., 2019). Conversely, it has been speculated that one reason why eukaryotes including yeast undergo genome-wide pervasive transcription is to allow RNA polymerase to survey the genome for replisome blocking lesions for repair by TC-NER prior to DNA replication (Ljungman, 2022), and direct evidence exists for this in bacteria as well as for telomeres in budding yeast (Guintini et al., 2022; Martinez et al., 2022). Furthermore, a yeast mutant deficient in global NER but proficient for TC-NER shows increased UV resistance when spurious transcription initiation is licensed, demonstrating the capacity of pervasive transcription to improve genome stability (Selvam et al., 2022).
Transcription therefore has an innately strand biased effect on genome stability, increasing damage on the non-template strand particularly during mutagen exposure, but resolving lesions on the template strand. Transcription does engage repair enzymes that introduce errors and strand breaks which may be mutagenic if encountered by the replisome.