Abstract
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.
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Acknowledgments
We thank Olga Vydzhak and Kristi Jensen for comments on the manuscript. BL’s lab is supported by the Heisenberg Program of the DFG-LU1709/2-1.
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Wagner, C.B., Luke, B. (2022). DNA–RNA Hybrids at Telomeres in Budding Yeast. In: Aguilera, A., Ruzov, A. (eds) R-Loops . Methods in Molecular Biology, vol 2528. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2477-7_10
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DOI: https://doi.org/10.1007/978-1-0716-2477-7_10
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