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.