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.