R-loops as a threat to genome stability
Transcription can result in the formation of R-loops, in which the newly
synthesised RNA hybridises with the DNA template strand prior to
re-annealing of the transcription bubble to form a stable DNA-RNA hybrid
structure (Figure 1A)(reviewed in (Santos-Pereira and Aguilera,
2015)) . R loops have physiological functions in transcription
and in accurate chromosome segregation (Boque-Sastre et al., 2015;
Kabeche et al., 2018), however, the pathological accumulation of R-loops
has been repeatedly shown to increase genetic instability (reviewed in
(Brickner et al., 2022; Huertas and Aguilera, 2003; Neil et al., 2018)).
Genome-wide profiling shows that R-loops form more readily following RNA
polymerase II pausing near transcription starting sites or transcription
termination sites (TSS or TTS, respectively) but also form
stochastically, particularly with polymerase stalling or in regions of
high GC content, and formation is facilitated by negative supercoiling
accumulated behind RNA polymerase (Chan et al., 2014; El Hage et al.,
2010; Skourti-Stathaki et al., 2014; Skourti-Stathaki et al., 2011;
Wahba et al., 2016).
Budding yeast utilises various mechanisms to ameliorate the impact of
R-loops on genome stability. Firstly, coating of nascent mRNA with
processing and export factors including THO/TREX reduces R-loop
formation and replication fork slowing (Garcia-Rubio et al., 2018;
Gomez-Gonzalez et al., 2011; Luna et al., 2019; Wellinger et al., 2006),
with THO/TREX mutants having unstable genomes (Prado et al., 1997; San
Martin-Alonso et al., 2021; Selvam et al., 2014). Another RNA-binding
protein, Npl3, which is involved in mRNA splicing and export in yeast,
was shown to decrease R-loop dependent genomic instability and
replication stress, and physical proximity of genes to nuclear pores
which accelerates mRNA export was shown to reduce R-loop accumulation
(Gaillard et al., 2018; Garcia-Benitez et al., 2017; Santos-Pereira et
al., 2013). Secondly, helicases can unwind R-loops; the transcription
termination factor Sen1 has a particular role in preventing R-loop
formation during S phase, with Sen1 depletion causing accumulation of
both R-loops and the ɣH2A histone marker which indicates the concomitant
onset of DNA replication stress (Khurana and Oberdoerffer, 2015; Mischo
et al., 2011; San Martin-Alonso et al., 2021). Thirdly, both RNase H1
and RNase H2 can degrade the RNA in R-loops. Mutants lacking RNase H2
have a much greater impact on R-loop mediated genome instability
(O’Connell et al., 2015; Zimmer and Koshland, 2016), but RNase H1 has an
’on-demand’ activity at the critical time in S phase (Lockhart et al.,
2019). Fourthly, chromatin modifiers including Rtt109 and FACT counter
the formation of R-loops (Canas et al., 2022; Herrera-Moyano et al.,
2014).
This multi-layered defence against R-loop formation and persistence is
not surprising given the genome instability that results when cells with
increased R-loops undergo DNA replication. However, the mechanism by
which R-loops cause genome instability remains unclear as replicative
helicases can unwind RNA-DNA helices in vitro and replication
forks can overcome DNA–RNA hybrids formed in co-directional
transcription units (Garcia-Rubio et al., 2018; Hamperl et al., 2017;
Shin and Kelman, 2006). Moreover, hyperrecombination phenotypes caused
by R-loops in yeast depend on Histone H3S10 phosphorylation suggesting
an indirect mechanism involving chromatin (Garcia-Pichardo et al.,
2017). One possible explanation is that in head-on encounters the RNA
polymerase becomes trapped between the R-loop and the replisome,
particularly in the absence of Sen1; this could impair normal mechanisms
for removing the RNA polymerase holoenzyme from DNA in front of the
replisome and form a potent roadblock (Felipe-Abrio et al., 2015;
Zardoni et al., 2021). Alternatively, as noted above ssDNA is more prone
to base damage, which would therefore accumulate on the non-template
strand in transcriptional R-loops. However, experiments to address this
mechanism discovered lesions arising from the replication bubble but
there was no dependence on R-loops (Williams et al., 2023). Finally,
R-loops can be targeted by the TC-NER or global genome NER pathway,
resulting in cleavage and removal of the RNA:DNA duplex followed by gap
repair (Crossley et al., 2023; Sollier et al., 2014), which forms a
short-lived ssDNA gap that could in theory be processed into a DSB by
DNA replication.
Negative supercoiling behind RNA polymerase can also promote the
formation of G-quadruplexes - non-B form four-stranded nucleic acid
structures made between several guanosines, often stabilised by
monovalent cations (Selvam et al., 2014). These structures can form
throughout the S. cerevisiae genome and particularly at telomeres
though they are not required for telomere function (Esnault et al.,
2023; Skourti-Stathaki et al., 2014). Like R-loops, these structures
have been implicated in replication fork stalling and leading to genome
instability, and are removed by the helicase Pif1 (Lopes et al., 2011;
Paeschke et al., 2011; Piazza et al., 2010).
Interruption of replisome progression by R-loops and G-quadruplexes has
the potential to be recombinogenic, and may contribute to genetic
heterogeneity even in unperturbed cells in a transcription-dependent
manner. However, R-loops and G-quadruplexes are common – the fact that
these species can be detected in genome wide assays even in wild-type
yeast, indicates that at any given site they are present in a
significant fraction of cells. If each R-loop even delayed the
replisome, let alone caused a time-consuming and mutagenic repair
process, this would dramatically slow DNA replication irrespective of
mutational burden. In reality, even RNase H1/H2 double mutants in which
R loops are highly stabilised are viable and do not show a substantial
growth defect (Zhao et al., 2018), suggesting that at most only a tiny
fraction of R-loops actually perturb replisome progression.