Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric non-coding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. Importantly, the cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, , suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.Cancer cells overcome replicative senescence by activating telomerase or the alternative lengthening of telomeres (ALT) pathway (1-3). ALT is used in ~5-15% of all human Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript cancers and is prevalent in specific cancer types, including osteosarcoma and glioblastoma (4). Currently, there are no therapies specifically targeting ALT. ALT relies on recombination to elongate telomeres (3), but how the recombinogenic state of ALT telomeres is established remains elusive. In contrast to cancer cells defective for homologous recombination (HR) and susceptible to Poly(ADP-ribose) polymerase (PARP) inhibition (5, 6), ALT-positive cells are HR-proficient (7). Thus, the reliance of ALT on recombination raises an important question as to whether recombination can be exploited in ALT-positive cancers as a means for targeted therapy.Single-stranded DNA (ssDNA) coated by replication protein A (RPA) is a key intermediate in both DNA replication and HR (8). RPA transiently associates with telomeres during DNA replication, but is released from telomeres after S phase (9, 10). The release of RPA may be an important mechanism to suppress HR at telomeres. The association of RPA with telomeres in S phase is facilitated by TERRA, the telomere repeat-containing RNA, which is also present at telomeres during this period (9,(11)(12)(13). To investigate how ALT is established, we determined whether the association of TERRA with telomeres is altered in ALT cells. TERRA colocalized with the telomere-binding protein TRF2 in telomerasepositive HeLa cervical cancer cells ( fig. S1) (9). However, in both HeLa and telomerasepositive SJSA1 osteosarcoma cells ( fig. S24B), the number of TERRA foci declined from S phase to G2 ( Considering that RPA is released from telomeres in G2/M when TERRA is repressed by ATRX (9), we examined whether ATRX is required for the release of RPA. In HeLa cells, numerous small replication-associated RPA foci (type-A RPA foci) were detected in S phase (Fig. S7). As cells progressed from S to ...

Maintenance of telomeres requires both DNA replication and telomere ‘capping’ by shelterin. These two processes employ 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 efficiently prevent RPA binding to telomeric ssDNA. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, 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 reaccumulation 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 following DNA replication, and promote telomere capping to preserve genomic integrity.