Cells are constantly exposed to external and internal processes that threaten the integrity of their genetic information. Even small errors or physical defects in the DNA strands that store the instructions required for life can have wide-ranging consequences. In response, cells deploy a variety of molecular actors to repair these lesions before they cause issues.

R-loops are a particularly complex form of genetic damage that emerge when a molecule known as RNA inserts itself into the DNA helix. If left unaddressed, the resulting structure interferes with the machinery that allows cells to replicate or express their genes – potentially leading to serious harm for the organism. Indeed, R-loops are often present in genes linked to a variety of cancers and neurological disorders. Despite their importance, how R-loops are normally removed is still not fully understood.

To explore this question, Roy et al. focused on the Smc5/6 complex, a molecular machine found across the tree of life that can help repair structurally complex genetic lesions. The team tested its involvement in R-loop removal using yeast strains that tend to carry more of these defects.

Yeast cells genetically manipulated to lack functional Smc5/6 complexes accumulated toxic levels of R-loops, resulting in extreme growth defects. Further investigations showed that the complex particularly targeted R-loops forming in highly expressed genes, as well as in genetic sequences important for preserving DNA integrity. Finally, Roy et al. used biochemical assays to explore how the human Smc5/6 complex specifically recognizes R-loops and assists an enzyme called RNase H – which can degrade RNA – in removing them.

Taken together, these findings deepen our understanding of R-loop dynamics; going forward, they may also help explain why mutations in components of the Smc5/6 complex are associated with severe genetic disorders in humans.

The maintenance of genome stability is a primordial function that ensures the proper development and homeostasis of all living organisms (Negrini et al., 2010; Hanahan and Weinberg, 2011; Lengauer et al., 1997; Cifone and Fidler, 1981). Successful maintenance of genomic integrity requires constant monitoring and repair of DNA lesions because genomes are under constant attack from endogenous sources of DNA damage (Tubbs and Nussenzweig, 2017; Thada and Greenberg, 2022; Lindahl and Nyberg, 1972). One of the most common sources of endogenous DNA damage is the formation of RNA-DNA hybrid structures in chromosomes. During transcription, nascent RNA transcripts can re-anneal to complementary DNA strands producing an RNA-DNA hybrid and displace the template strand, creating an obstacle to the progression of the DNA replication machinery. The resulting RNA-DNA hybrid and displaced ssDNA segment —together termed the R-loop— are highly deleterious for genome integrity (Chatzidoukaki et al., 2021; Costantino and Koshland, 2018; De Magis et al., 2019; Stork et al., 2016). RNA-DNA hybrid structures can also be formed under a variety of physiological conditions in eukaryotic genomes and play important roles in cell physiology and regulate genome dynamics. Common physiological roles of RNA-DNA hybrids include immunoglobulin class switching recombination of B cells in vertebrates (Yu et al., 2003; Roy et al., 2008), mitochondrial DNA replication (Pohjoismäki et al., 2010; Xu and Clayton, 1996), bacterial plasmid replication (Baker and Kornberg, 1988; McLean et al., 2022), and CRISPR-Cas9 gene editing (Zhang et al., 2021; Xiao et al., 2017). Moreover, RNA-DNA hybrids co-ordinate specific regulatory steps in transcription initiation and termination (Sidorenkov et al., 1998; Skourti-Stathaki et al., 2011; Nudler et al., 1997), telomere homeostasis (Balk et al., 2013; Luke et al., 2008), and gene expression (Crossley et al., 2019; García-Muse and Aguilera, 2019; Brambati et al., 2020; Niehrs and Luke, 2020; Zardoni et al., 2021). However, the formation of unprogrammed or non-physiological RNA-DNA hybrid structures can interfere with DNA replication-related processes, resulting in replicative stress and the formation of DNA double-strand breaks (DSBs) (Crossley et al., 2019; Aguilera and García-Muse, 2012; Santos-Pereira and Aguilera, 2015; Hamperl et al., 2017; Kumar et al., 2021; Kim et al., 2024). Importantly, exposed ssDNA in the R-loop can be cleaved by different endonucleases leading to DNA breaks and/or mutagenic events, and can also adopt harmful secondary structures (Freudenreich, 2018; Miglietta et al., 2020). R-loop-induced DNA damage and genomic rearrangements have been linked to various disease states in humans. Examples include trinucleotide repeat-associated diseases, auto-immune disorders, neurological disorders, and cancer, although it is not currently known whether R-loops play a causative or consequential role in such diseases (García-Muse and Aguilera, 2019; Richard and Manley, 2017).

To mitigate the toxic consequences associated with the presence of unprogrammed RNA-DNA hybrids in chromosomes, several cellular mechanisms work in concert to prevent their formation, and when they do accumulate, remove them from eukaryotic genomes. For instance, the Ribonuclease H (RNase H) family of enzymes plays a central role in degrading the RNA moiety of R-loops created under a variety of genomic conditions (Lazzaro et al., 2012; Lockhart et al., 2019; Figure 1A). Recently, it was shown that the RNase DICER can also cleave the RNA moiety of the R-loops in higher eukaryotes (Camino et al., 2023). In addition, factors associated with transcription and mRNA biogenesis, RNA-DNA helicases, topoisomerases, chromatin remodelers, and several DNA repair enzymes are known to be involved alongside RNase H in preventing the accumulation of R-loop structures in eukaryotic chromosomes (García-Muse and Aguilera, 2019). However, a definitive understanding of the sequence of events and exact molecular mechanisms responsible for the repair of R-loops/RNA-DNA hybrids remains to be established.

Figure 1 with 1 supplement see all

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Work from our laboratory and other researchers provides hints of a possible involvement of structural maintenance of chromosomes (SMC)-type complexes in RNA-DNA hybrid metabolism (Lafuente-Barquero et al., 2017; Serrano et al., 2020; Girasol et al., 2023; Penzo et al., 2023). SMC complexes are effectors of large-scale changes in chromosome organization and include three evolutionarily conserved enzyme complexes: condensin, cohesin, and the Smc5/6 complex (Peng and Zhao, 2023). The Smc5/6 complex is a particularly intriguing member of this family because its function —unlike that of cohesin and condensin— is primarily concerned with DNA repair, and yet its exact contribution to this process is not fully understood. Recent evidence suggests that the Smc5/6 complex is a DNA compacting enzyme that acts in vivo by regulating local chromatin domains containing unusual DNA structures that can lead to replication stress and/or DNA damage (Serrano et al., 2020; Gutierrez-Escribano et al., 2020; Tanasie et al., 2022; Pradhan et al., 2023). Interestingly, genome-wide screens as well as targeted genetic analyses revealed synthetic interactions among a subset of mutants affecting Smc5/6 complex components and RNA-DNA hybrid detoxification enzymes (Lafuente-Barquero et al., 2017; Costanzo et al., 2016; Styles et al., 2016; Kuzmin et al., 2018; Chang et al., 2019). Consistent with this, we have previously shown that the Smc5/6 complex can bind to short RNA-DNA duplexes with high affinity and specificity (Serrano et al., 2020). Together, these results raise the intriguing possibility that the Smc5/6 complex might be involved in the detection and/or processing of toxic R-loops formed in eukaryotic genomes.

Here, we show that the Smc5/6 complex is directly involved in the repair of unscheduled R-loops generated by a diverse set of genomic transactions. In particular, we demonstrate that the Smc5/6 complex binds strongly to R-loop structures formed during active gene transcription and promotes RNase H2-mediated degradation of the RNA component of R-loops. Our results unravel a hitherto unanticipated role for the Smc5/6 complex in the removal of RNA structures from chromosomes, an essential function for the maintenance of genome integrity and cell fitness.

The incorporation of RNA in chromosomal DNA represents a unique challenge for the stability of eukaryotic genomes because this modification can occur in both physiological and pathogenic conditions. Maintaining a finely balanced cycle of R-loop formation and removal in chromosomes is crucial for the overall fitness of cells because altered RNA-DNA hybrid homeostasis can result in DNA damage and genomic instability, ultimately contributing to the development of several pathological conditions (Mackay et al., 2020; Crossley et al., 2023). Here, we show that the Smc5/6 complex promotes the removal of toxic R-loops in eukaryotic chromosomes. While previous genetic experiments have supported a role for the Smc5/6 complex in the natural regulation of TERRA levels at telomeres (Lafuente-Barquero et al., 2017; Moradi-Fard et al., 2016), our study reports the first demonstration that Smc5/6 complex activity is essential for the removal of unscheduled R-loops from the genome. This discovery is significant because non-physiological R-loops represent the most toxic and damaging source of RNA-DNA hybrids for genome stability (Crossley et al., 2019; Brambati et al., 2020; Petermann et al., 2022) and failure to remove these structures exacts a heavy toll on cell fitness. Moreover, we demonstrate for the first time that the Smc5/6 complex can directly recognize R-loops, suggesting an early role in the detection and repair of these structures in vivo (see model in Figure 7). Consistent with this suggestion, the Smc5/6 complex has been shown by chromatin immunoprecipitation to accumulate at sites that are common R-loop enrichment zones on chromosomes, including the rDNA locus, telomeres, and highly transcribed/difficult-to-replicate chromosomal loci (Diman et al., 2023; Jeppsson et al., 2023; Jeppsson et al., 2014; Pebernard et al., 2008). We showed that inactivation of Smc5/6 components leads to an increase in R-loop formation at several of these loci in the absence of RNase H enzyme activity, a result that aligns nicely with Smc5/6 complex localization in live cells.

Figure 7

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Our genetic enhancement results obtained with double mutants must be interpreted carefully because they involve conditional/hypomorphic alleles of Smc5/6 complex components. Synthetic or enhancement phenotypes involving non-null alleles reflect the contribution of two mutations to the same cellular process, but not necessarily or exclusively in the same molecular pathway (i.e., interactions ‘within pathways’ and ‘between pathways;’ reviewed in Huang and Sternberg, 1995; Boone et al., 2007; Roth et al., 2009). As such, the exacerbation of the DNA damage sensitivity of RNase H mutants by temperature-sensitive alleles of the Smc5/6 complex may be the consequence of their effects on RNA-DNA hybrid removal (i.e., thus reflecting a ‘within pathway’ contribution relative to RNase H1/H2) and their roles in additional biochemical pathways distinct from RNA-DNA hybrid degradation but still relevant to R-loop detoxification. We favor a model where the Smc5/6 complex acts at two distinct levels –within and between pathways– in the cellular response to R-loop formation (Figure 7). First, Smc5/6 contributes to RNase H-dependent removal of RNA-DNA hybrids from genomic DNA, as shown in Figures 1 and 2. In the execution of this function, the Smc5/6 role is substantial but not as extensive as that of RNase H enzymes (more on this below). Second, the Smc5/6 complex plays an important role in the maintenance of DNA replication fork stability, as previously established (Peng and Zhao, 2023). This role is crucial for cellular fitness in the presence of elevated R-loop levels because these structures often disrupt replication fork progression and can lead to fork collapse (Crossley et al., 2019; Brambati et al., 2020; Petermann et al., 2022; Kemiha et al., 2021). In this context, losing both RNase H and Smc5/6 complex activities will have consequences well beyond those observed in individual mutants because it will increase R-loop formation in a context where cells have lost the ability to cope with stress at replication forks. This ‘dual hit’ will render cells hypersensitive to R-loops, thus explaining the synthetic effects of combining smc5/6 and rnase H mutations. Interestingly, the dual hit scenario leading to additive phenotypes appears to be the prevalent paradigm observed with effectors of RNA-DNA hybrid metabolism. For instance, past genetic interaction studies have shown that combining sen1-1 or sen1-3 alleles with rnh1Δ rnh201Δ mutations leads to synthetic lethality (Appanah et al., 2020). Likewise, inactivation of the THO complex induces a substantial increase in RNA-DNA hybrids in cells defective in RNase H activity (Yang et al., 2021), similar to our observation with nse4-4 rnh1Δ rnh201Δ and smc6-9 rnh1Δ rnh201Δ mutant strains (Figure 2). This pattern of synthetic enhancements when inactivating effectors of RNA-DNA hybrid metabolism is consistent with multiple mechanisms acting independently to promote the removal of toxic or unscheduled R-loops from eukaryotic genomes.

How might the Smc5/6 complex association with R-loops promote their degradation in vivo? Hints of a potential mechanism of action come from the observation that one of the enzymes responsible for R-loop degradation, RNase H1, shows little enzymatic activity under basal conditions and requires stimulation by ancillary factors, such as RPA, to achieve maximal R-loop degradation (Nguyen et al., 2017). It therefore seems plausible that RNase H2 might also require the assistance of a separate stimulatory factor to achieve maximal catalytic efficiency. Testing this notion in a reconstituted R-loop degradation assay confirmed the model that RNase H2 activity can be stimulated effectively, and in a dose-dependent manner by the Smc5/6 complex. The two RNase H enzymes differ, however, in that RNase H1 shows very little RNA degradation activity in the absence of RPA (Nguyen et al., 2017), whereas RNase H2 is moderately active as an R-loop degrading enzyme at basal state (Figure 6 and Figure 6—figure supplement 1). The implication for the Smc5/6 complex is that it is probably not required to stimulate RNase H2 activity in all contexts in vivo but is likely more important in challenging environments where R-loops are highly abundant or otherwise difficult to degrade effectively by RNase H2 alone. This interpretation dovetails nicely with the synthetic interaction profiles we observed when combining Smc5/6 complex mutations with mutants that increase R-loop formation at selected genomic locations (Figure 4). Taken together, our genetic analyses indicate that the repertoire of genomic lesions that are substrates for RNase H2 and the Smc5/6 complex in vivo is not fully overlapping.

An RNase H2 stimulatory role for the Smc5/6 complex is compelling because it provides a cellular capacity/buffer to address substantial fluctuations in the total load of RNA-DNA hybrids produced under physiological and non-physiological conditions (Mackay et al., 2020; Petermann et al., 2022). Consequently, reducing the total load of RNA-DNA hybrids or altering its sources of origin is expected to modify the requirement for the Smc5/6 complex in R-loop metabolism (Lafuente-Barquero et al., 2017). How might Smc5/6 stimulate RNase H2 enzymatic activity? This is a question for a future study, but it has not escaped our attention that RNase H1 is strongly stimulated by a ssDNA binding protein, RPA (Nguyen et al., 2017), a biochemical property also encoded in the Smc5/6 complex (Serrano et al., 2020; Roy and D’Amours, 2011; Roy et al., 2011; Roy et al., 2015). Separate from this possibility, the Smc5/6 complex plays a vital role in promoting RPA binding and maintenance at ssDNA during homologous recombination (Tanasie et al., 2022), a function that could indirectly stimulate RNase H1 activity and R-loop repair (Nguyen et al., 2017). Addressing these possibilities will require the identification of mutations abrogating the ssDNA binding activity of the Smc5/6 complex, a difficult feat for a holoenzyme known to associate with DNA through multiple different binding modes and domains (i.e. topological and electrostatic; Serrano et al., 2020; Roy et al., 2015).

Up to now, the Smc5/6 complex has been thought of primarily as a genome stability factor associated with the repair of DSBs, recovery of stalled replication forks, telomeric length maintenance, and virus restriction (Peng and Zhao, 2023). While it is not evident why the Smc5/6 complex would be involved in such a diverse and loosely connected group of cellular functions, it is nevertheless clear that failure to execute these functions generates toxic recombination intermediates in vivo. Under normal circumstances, the Smc5/6 complex binds to ssDNA intermediates and branched/structured substrates at DNA repair sites and the association of the complex to these DNA intermediates provides a platform for repair factors to resolve toxic DNA lesions (Serrano et al., 2020; Gutierrez-Escribano et al., 2020; Tanasie et al., 2022). It is interesting to note that most of the nuclear processes outlined above involve the formation of RNA-DNA hybrids in one form or another (Brambati et al., 2020; Petermann et al., 2022). As such, the function we uncovered for the Smc5/6 complex in RNA-DNA metabolism may be a unifying role that explains its involvement in such a diverse repertoire of cellular functions. More work will be required to test this exciting possibility.

In conclusion, our work demonstrates a direct and active involvement of the Smc5/6 complex in the removal of R-loops from eukaryotic chromosomes. We showed the Smc5/6 complex binds strongly to RNA-DNA hybrid structures formed during active gene transcription and telomere length regulation, and subsequently promotes the removal of these toxic structures via the stimulation of RNase H2 enzymatic activity. This work uncovered a previously unanticipated contribution of the Smc5/6 complex in genome stability with important ramifications for the health and disease of all eukaryotic organisms.

All source Data files have been provided for Figures 2-3, Figure 5-6, and Figure supplements on the Dryad open repository site at https://doi.org/10.5061/dryad.3xsj3txpg.

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Author details

1. Shamayita Roy

   Ottawa Institute of Systems Biology, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0001-9732-1446

2. Hemanta Adhikary

   Ottawa Institute of Systems Biology, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada

   Contribution

   Formal analysis, Validation, Investigation, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Sarah Isler

   Ottawa Institute of Systems Biology, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada

   Contribution

   Formal analysis, Validation, Investigation, Writing – review and editing, Sarah Isler has been added as an author at the revised stage. She has been added at this stage to perform some experiments suggested by the reviewers. We confirm that all authors agree with their inclusion and place in the author list

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0005-2470-2327

4. Damien D'Amours

   Ottawa Institute of Systems Biology, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   damien.damours@uottawa.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2183-9951

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