IntroductionReplication stress can contribute to tumorigenesis via genome instability [1] but is also a therapeutic target and a central mechanism of action for many cytotoxic cancer therapies [2]. Nuclear processes such as transcription can contribute to replication stress by generating barriers to replication fork progression. Transcription can interfere with replication fork progression through topological stress, collisions of the RNA and DNA polymerase machineries or co-transcriptional RNA:DNA hybrids called R-loops [3].Oncogenes such as HRAS, SS18-SSX1 and EWS–FLI1 increase RNA:DNA hybrid levels and transcription-replication conflicts (TRCs) [4,5,6]. Chemotherapies can also induce RNA:DNA hybrids. For instance, camptothecins (CPT) inhibit DNA topoisomerase I (TOP1), which alleviates supercoiling, increasing the likelihood of R-loop formation [7]. Gemcitabine and hydroxyurea deplete deoxyribonucleotides which can lead to increased ribonucleotide incorporation [8, 9] and stalled replication forks, which can in turn promote R-loop formation [10]. RNA:DNA hybrids can thereby contribute to DNA damage, genome instability and cell death caused by chemotherapies. They may also contribute to the toxicity of targeted cancer therapies such as ATR inhibitors [11] and to innate immune activation [12].R-loop homoeostasis is a fine balance between R-loop formation and removal. In eukaryotes, RNase H1 and RNase H2 can digest the RNA component of RNA:DNA hybrids [13]. RNase H2 is a heterotrimer where RNASEH2A contains the catalytic domain, whereas RNASEH2B and RNASEH2C are essential accessory subunits [14, 15]. RNASEH2B is required for the nuclear localisation of the RNase H2 complex and interacts with the DNA sliding clamp, PCNA [16, 17]. RNase H2 initiates ribonucleotide excision repair (RER), the removal of mis-incorporated single ribonucleotides from duplex DNA [18] and can remove R-loops (Fig. 1A). RNase H2 is the predominant RNase H activity in yeast and mammals [19, 20]. In S. cerevisiae RNase H2 protein levels are cell-cycle regulated [21, 22] and R-loops at replication forks are thought to be processed specifically by RNase H2 [23]. However, in mammals RNase H2 levels appear to be constant throughout the cell cycle [8, 9] and the contribution of this enzyme to R-loop removal is less well studied. RNase H2 deficiency can cause neuroinflammation [24] or contribute to cancer development [25, 26], whereas RNase H2 upregulation is associated with cancer progression [27,28,29]. R-loop interacting proteins such as RNase H2 are potential therapeutic targets in cancer [29].Fig. 1: Oncogene-induced replication stress increases RNase H2 protein levels and -activity.A RNase H2 complex subunits and functions. B Protein levels of HRAS, RNASEH2B (RH2B), RNASEH2C (RH2C) and Vinculin (loading control) in BJ-hTERT HRASV12ERTAM cells ± 72 h HRASG12V induction with 4-hydroxytamoxifen (4-OHT). C Relative protein levels of RH2B and RH2C after 24, 72 or 96 h HRASG12V induction normalised to ethanol (con) for each protein. N = 5 (24 and 96 h), N = 13 (RH2B 72 h), N = 6 (RH2C 72 h). D RT-qPCR analysis of RNASEH2A, RNASEH2B and RNASEH2C expression after HRASG12V induction. Asterisks compare to con. N = 4. E Substrates used for the RNase H2 activity assay with forward strand covalently coupled to 3′-fluorescein (green) and reverse strand linked to 5′-DABCYL quencher (orange). F Representative time course of DRD:DNA substrate conversion during incubation with whole cell extract ±72 h HRASG12V induction (control). N = 2 measured on the same plate. G Relative RNase H2 activities in cell extracts ±72 h HRASG12V induction. N = 4. H Protein levels of CYCLIN E, RH2A, RH2B, RH2C and Tubulin (loading control) in U2OS-CYCLIN E cells ± CYCLIN E induction with tetracycline removal (-tet) for the times indicated. I Protein levels of RH2A, RH2B, RH2C and Tubulin (loading control) in BJ-hTERT cells after 4 h treatment with 200 μM hydroxyurea (HU), 25 nM gemcitabine (GEM), 10 μM camptothecin (CPT) or DMSO (con). Means and SEM (bars) of independent experiments are shown. Asterisks indicate p values (ANOVA or mixed-effects analysis, *p