RNA-DNA hybrids prevent resection at dysfunctional telomeres.

At critically short telomeres, stabilized TERRA RNA-DNA hybrids drive homology-directed repair (HDR) to delay replicative senescence. However, even at long- and intermediate-length telomeres, not subject to HDR, transient TERRA RNA-DNA hybrids form, suggestive of additional roles. We report that telomeric RNA-DNA hybrids prevent Exo1-mediated resection when telomeres become non-functional. We used the well-characterized cdc13-1 allele, where telomere resection can be induced in a temperature-dependent manner, to demonstrate that ssDNA generation at telomeres is either prevented or augmented when RNA-DNA hybrids are stabilized or destabilized, respectively. The viability of cdc13-1 cells is affected by the presence or absence of hybrids accordingly. Telomeric hybrids do not affect the shortening rate of bulk telomeres. We suggest that TERRA hybrids require dynamic regulation to drive HDR at short telomeres; hybrid presence may initiate HDR through replication stress, whereby their removal allows strand resection.

TERRA increases at short telomeres in yeast survivors and regulates survivor associated senescence (SAS).

Cancer cells achieve immortality by employing either homology-directed repair (HDR) or the telomerase enzyme to maintain telomeres. ALT (alternative lengthening of telomeres) refers to the subset of cancer cells that employ HDR. Many ALT features are conserved from yeast to human cells, with the yeast equivalent being referred to as survivors. The non-coding RNA TERRA, and its ability to form RNA-DNA hybrids, has been implicated in ALT/survivor maintenance by promoting HDR. It is not understood which telomeres in ALT/survivors engage in HDR, nor is it clear which telomeres upregulate TERRA. Using yeast survivors as a model for ALT, we demonstrate that HDR only occurs at telomeres when they become critically short. Moreover, TERRA levels steadily increase as telomeres shorten and decrease again following HDR-mediated recombination. We observe that survivors undergo cycles of senescence, in a similar manner to non-survivors following telomerase loss, which we refer to as survivor associated senescence (SAS). Similar to 'normal' senescence, we report that RNA-DNA hybrids slow the rate of SAS, likely through the elongation of critically short telomeres, however decreasing the rate of telomere shortening may contribute to this effect. In summary, TERRA RNA-DNA hybrids regulate telomere dysfunction-induced senescence before and after survivor formation.

DNA-RNA Hybrids at Telomeres in Budding Yeast.

It has recently been demonstrated that budding yeast telomeres are transcribed into TERRA, a long noncoding RNA. Due to the G-rich nature of the coding strand, TERRA has a tendency to form DNA-RNA hybrids and potentially R-loops, which in turn, promote repair at short telomeres. Here, we report two methods to detect DNA-RNA hybrids at yeast telomeres, namely, DRIP, which employs the S9.6 hybrid-recognizing antibody, and R-ChIP, which takes advantage of a catalytic dead form of RNase H1 (Rnh1-cd). We use cross-linked material for both protocols as we have found that this does not negatively affect recovered material, and furthermore allows the precipitation of other proteins from the identical cross-linked material. Although both methods are successful in terms of detecting DNA-RNA hybrids at telomeres, the R-ChIP method yields an approximately ten-fold increased enrichment.