TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA

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Abstract
Two single-stranded DNA binding proteins, POT1 and RPA, associate with the ends of DNA chromosomes, which are known as telomeres. Binding of RPA to telomeres can activate a DNA damage response, so it was suggested that POT1 might bind telomeres to prevent RPA association. Zou and colleagues unexpectedly find that POT1 and its partner TPP1 do not prevent RPA binding, but hnRNPA1 does. TERRA, a telomere-associated RNA, displaces hnRNPA1 and promotes POT1 binding after S phase, when replication is completed. Two single-stranded DNA-binding proteins, POT1 and RPA, associate with telomeres. Binding of RPA to telomeres can activate a DNA damage response, so it was previously proposed that POT1 binds telomeres to prevent RPA association. Here, it is found that POT1–TPP1 cannot prevent RPA binding, and hnRNPA1 is identified as having this activity instead. In addition, it is shown that TERRA, a telomere-associated RNA, displaces hnRNPA1 and promotes POT1 binding after S phase, when replication is completed. Maintenance of telomeres requires both DNA replication and telomere ‘capping’ by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres1,2, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres3. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.