Known examples of transcription-induced genetic change
Effective DNA repair of spontaneous damage and the excellent processivity of the replisome form a multi-layered defence against potential mutations despite the considerable strain imposed by transcription. In keeping with this, mutation accumulation studies find only minimal evidence of a link between mutation and transcription (Chen and Zhang, 2014; Zhu et al., 2014), but clear examples of transcription-induced mutation have been reported and mechanistic drivers have been elucidated.
We suggest that stalling of DNA replication forks either spontaneously or due to a replication-transcription conflict will occasionally require fork restart by BIR, but de novo mutation only arises when poorly processive BIR forks encounter obstacles resulting (for example) from transcription (illustrated in Figure 2A,B). In other words, two separable events are required for a mutagenic outcome. Direct measurements of BIR fork progression reveal them to be profoundly impaired and prone to template switching by head-on encounters with transcription, as well as epigenetic marks to which the replisome is insensitive (Che et al., 2015; Liu et al., 2021).
The best characterised example of transcription-induced recombination in yeast occurs at the ribosomal DNA. Under the standard model, replication forks stalled at the ribosomal DNA RFB are cleaved, resected then undergo non-allelic homologous recombination to cause CNV if cohesin is removed by the transcription of non-coding RNAs to facilitate recombination between repeats (Kobayashi and Ganley, 2005; Kobayashi et al., 2004). However, replication forks are stably paused at the RFB and it is unclear why these would be cleaved prior to resolution by oncoming replication forks (Carr and Lambert, 2013). Nonetheless, rDNA hyper-recombination occurs in mutants lacking polymerase component Pol32 or deacetylase Hst3/Hst4, which should only impact the processivity of BIR forks, indicating that BIR is involved (Che et al., 2015; Houseley and Tollervey, 2011; Ide et al., 2013; Jack et al., 2015). Replication forks paused at the RFB are stable (Calzada et al., 2005), but the prominent double strand break signal from the RFB indicates that replication fork reversal occurs, so resection and reinvasion of this end is likely to occur at some frequency (Burkhalter and Sogo, 2004; Kara et al., 2021; Zhu et al., 2019). The BIR events initiated are likely short-lived due to resolution by oncoming forks, but could easily encounter oncoming RNA polymerase I and be forced into template switching. This would require the BIR fork to traverse the RFB, but given that the RFB is mediated by FPC which is bound to CMG and that fork reversal dissociates the polymerases from CMG, this is plausible (Figure 2C).
Recently, we demonstrated that copper resistance acquisition in yeast arises through transcriptionally induced copy number amplification of the CUP1 gene (Hull et al., 2017; Whale et al., 2022). The mechanism proved extremely complex, requiring a BIR event that becomes error-prone on encountering an epigenetic scar at a low-efficiency replication origin upstream of the gene itself. Transcriptional dependence requires the activator complex Mediator and the mRNA export complex TREX-2 which are recruited during activation of inducible genes such as CUP1 . The only detectable TREX-2-dependent replication fork stalling at the locus occurred at the inefficient replication origin, suggesting that the primary impact of transcription is to prevent the successful firing of this origin. The unsuccessful origin firing leaves a scar which causes BIR forks repairing local replisome stalling to undergo template switching, resulting in copy number amplification (Figure 2D). Importantly, this behaviour was not specific to the CUP1 gene as the SFA1 gene, which bestows formaldehyde resistance and like CUP1 has an inefficient replication origin just upstream, can undergo an equivalent transcriptionally-induced gene amplification through BIR (Hull et al., 2017).
It is worth noting that genetic changes induced by both these systems are not restricted to chromosomes. Both ribosomal DNA and CUP1loci are susceptible to transcription-induced extrachromosomal circular DNA (eccDNA)  formation. These species, particularly extrachromosomal ribosomal DNA circles (ERCs), accumulate to massive levels in aged cells, adding 30-40% to genome size (Cruz et al., 2018; Hull et al., 2019; Sinclair and Guarente, 1997), and ability of eccDNA to reintegrate in chromosomal DNA provides an additional pathway by which major transcription-induced genetic changes can arise (Beverley et al., 1984; Brewer et al., 2015; Demeke et al., 2015; Galeote et al., 2011)
Interpretation of many studies in this area is complex due to the use of the extraordinarily highly expressed GAL1 promoter; which is one of the few for which we can detect transcription-induced pausing of the replisome by TrAEL-seq (Kara et al., 2021). This may be sufficient to directly induce local BIR events at some frequency, and the pattern of transcription-induced mutations induced in a lys2 reporter are consistent with those reported to occur during BIR (Datta and Jinks-Robertson, 1995). However, whether those mutations arise through conflicts between the BIR fork and transcription is hard to resolve if the rate of BIR induction is not necessarily constant, though follow-on studies showed a direct correlation between transcriptional strength and reversion of lys2 mutations, as well as induction of recombination events (Kim et al., 2007; Saxe et al., 2000), all consistent with BIR.
Overall, where it is possible to separate the individual contributions of direct impacts of transcription on the replisome and on BIR, the evidence is consistent with mechanisms in which transcription-replisome conflicts are at most the initiator, whereas the actual mutations are caused by further conflicts impairing the processivity of BIR. We suggest that the danger posed by increased sensitivity of BIR forks to transcription-related obstacles is the reason why transcription is globally down-regulated in response to DNA replication stress (reviewed in (Silva and Ideker, 2019)). Notably however, for the ribosomal DNA,CUP1 and SFA1 transcription-induced genomic instability relies on specific properties of the locus, raising the question of whether some genes are configured in a manner that might be more prone to mutation or genome rearrangement.