R-loops at the telomere as a toxic source of genomic instability

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Abstract Damage to telomeres resulting from radiation or exposure to toxic chemicals can lead to cancer and accelerated aging due to unwanted telomere shortening. Damage may also result from normal processes including generation of formaldehyde or transcription when RNA is left behind embedded in the DNA in the form of R-loops. Extensive studies of genomic R-loops have shown them to play both positive roles in regulation of transcription and harmful roles leading to DNA breakage and mutagenesis. Telomeric R-loops (t- R-loops) may possibly be the single greatest source of DNA damage at telomeres. T-R-loops occur in normal human cells and are more abundant in ALT cancer cells and cells mutated in certain DNA methylases that result in high levels of telomeric RNA (TERRA). Elevated levels of t-R-loops have been linked to telomere damage and shortening. Radiation, toxic agents such as cisplatin, formaldehyde, and exposure to oxidative damage are also likely to generate higher levels of t-R-loops. We demonstrated that telomeres are arranged in large loops (t-loops) and recently made a paradigm-shifting discovery linking t-loop formation to telomere transcription which generates TERRA and produces t-R-loops which we propose are key to t-loop formation. Thus, telomeric R-loops may be both toxic and necessary for forming protective t-loops. T-R-loops are more stable than normal R-loops due to G-quartet formation. The extensive studies of genomic and telomeric R- loops have all relied on a single assay employing the highly specific S9.6 antibody to DNA/RNA hybrids (DRIP assay). While having driven the field, this IP assay does not discriminate between one or many R-loops on a DNA fragment or provide information on the clustering of the R-loops, or their size. For t-R-loops, the IP assay does not reveal whether there are R-loops within the looped portion of the t-loop or their distribution from the sub-telomeric regions to the telomere terminus. For the field to progress, such critical information must be obtained. This can now be done using direct electron microscopic (EM) visualization using methods we have verified in a plasmid-based R-loop study. In our proposed work we carry out a high-resolution study of the large (120-240 nt) particles formed by single stranded G-rich telomeric DNA and we propose, TERRA RNA. This is critical for understanding the structure of t-R-loops and will be done cryoEM. To directly determine the frequency, location, size and clustering of t-R-loops we will apply a novel affinity isolation for telomeric DNA, combined with a battery of EM tools. This will be done using cultured HeLa and human ALT cancer lines and extended to cells treated with toxic chemicals including cisplatin and formaldehyde to introduce crosslinks in the DNA. A novel chemoptogenomic approach for placing ROS generated 8-oxo-G lesions specifically at the telomere in cells will be applied in a collaboration and the result on the levels of t-R-loops determined.