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Novel data indicate that human RNase H1 physically and functionally interacts with the trimeric single-stranded DNA binding protein complex RPA (Nguyen et al., 2017b). The interaction with RPA stimulates RNase H1 binding to R-loops both in vitro and in vivo, and additionally it stimulates RNase H1 catalytic activity on R-loops in vitro. Indeed, RPA and RNase H1 colocalize at R-loops in cells. In RNase H1 mutants that retain R-loop resolution activity in vitro but are compromised in RPA binding, RNase H1 fails to localize to R-loops and cannot process them in vivo. Furthermore, when overexpressed, mutant RNase H1 cannot rescue the R-loop mediated genome instability in R-loop accumulating mutants, whereas overexpression of wild type RNase H1 can (Nguyen et al., 2017b). These data indicate that RPA is an R-loop sensor and that the interaction between RPA and RNase H1 is important for the ability of RNase H1 to bind R-loops and to promote their degradation; interestingly, no interaction between RPA and RNase H2 has been detected in this study (Nguyen et al., 2017b), suggesting that RPA might not be required for RNase H2 recruitment to R-loops. The RPA-RNase H1 interaction is conserved in bacteria, as in E. coli the single-stranded DNA-binding protein (SSB) binds to RNase HI in vivo and stimulates its activity on R-loops in vitro (Petzold et al., 2015). Moreover, in S. cerevisiae RNase H1 was found to interact with two subunits of the RPA complex, namely Rfa1 and Rfa3, by a proteomic approach (Gavin et al., 2002).

The functional interaction between RPA and RNase H1 could therefore be a conserved mechanism in place to recruit RNase H1 to R-loops. Importantly, this interaction could be relevant for the recruitment of RNase H1 to short telomeres: R-loop stabilization at short telomeres due to local RNase H2 depletion might lead to increased recruitment of RPA to the displaced single stranded filament; indeed, RPA localizes to stabilized R-loops in human cells (Nguyen et al., 2017b). RPA binding to stabilized telomeric R-loops might recruit RNase H1 and promote its activity. Furthermore, the exonucleolytic processing of the 5’ C-strand (the one involved in the R-loop) to allow HR initiation, would lead to further RPA accumulation on the telomeric 3’ overhang, and thereby enhanced local RNase H1 recruitment/activation. Importantly, short telomeres possess long ssDNA overhangs generated by resection activities (Fallet et al., 2014)(Figure 25). This RPA-mediated RNase H1 recruitment might be required for efficient resection of the C-strand (Ohle et

al., 2016) and promotion of HR by generating a long recombinogenic single stranded DNA. In line with this, RNase H1 depletion in telomerase negative cells leads to accelerated senescence onset, which could be due to impaired HR at these telomeres. Interestingly, RPA accumulates at telomeres in ALT cancer cells and seems to be regulated by ATRX activity, as upon ATRX knockdown RPA accumulates at ALT telomeres in G2 phase, which is when TERRA foci at telomeres are stabilized (Flynn et al., 2015; O'Sullivan et al., 2014). Therefore, RPA increased presence at ALT telomeres, which may be due to increased R-loop presence, might be what allows RNase H1 binding exclusively at ALT telomeres and not at telomeres in normal cells.

Figure 25. Possible explanation for RNase H1 recruitment to short telomeres.

The single stranded component of stabilized R-loops at short telomeres might be bound by RPA. After R-loop mediated induction of replication stress, resection of the 5’ end ensues and recruits more RPA to the opposite strand. Local increased RPA concentration might promote RNase H1 recruitment and activity to remove the R-loop, thereby allowing efficient resection and subsequent BIR-mediated recombination.

We can therefore propose the following model (Figure 26). At long telomeres, RNase H2 has a major role in regulating the timing of R-loops removal, in order to avoid collisions with the replication machinery; RNase H1 might play a compensatory role if RNase H2 is impaired, which could explain why only the double mutant rnh1 rnh201 displays increased R-loop levels (observation by A. Maicher). As telomeres shorten and RNase H2 recruitment to telomeres is reduced, RNase H1 instead localizes to telomeres; RNase H1 localization and activity might be promoted by RPA enrichment at short telomeres, and may allow efficient resection of the 5’-strand. This model would explain the different senescence kinetics of cells depleted for RNase H enzymes. In the absence of RNase H1, critically short telomeres accumulating R-loops may be impaired in the resection of the 5’ strand, leading to impaired recombination and therefore faster senescence. In the absence of RNase H2, the timing of R-loop removal at long telomeres might be impaired, although RNase H1 might still be able to partially remove them. This might lead to recombination already at long telomeres and following efficient resection of the C-strand, therefore causing delayed senescence onset. In the double mutant, R-loop deregulation at long telomeres will promote early recombination, which might be resected inefficiently due to stabilization of the R-loop which might render the 5’ strand less accessible to exonucleases, and therefore result in an intermediate phenotype of senescence kinetics. On the contrary, in the case of RNase H1 overexpression, degradation of telomeric R- loops at all steps will impair telomere recombination and lead to fast senescence.

Figure 26. RNase H1 and H2 might functions at telomeres in different steps.

At long telomeres, Rif2 recruits RNase H2 to remove R-loops before the arrival of the replication fork to telomeres. RNase H1 might act at long telomeres when RNase H2 is impaired. As telomeres shorten, RNase H2 loss allows R-loop accumulation and consequent replication stress; RPA might bind to the R-loop. RNase H1 might be recruited to short telomeres by increased localization of RPA, and promote efficient 5’ end resection, which initiates BIR-dependent telomere elongation.