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

Abstract

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.

PTEN regulates RPA1 and protects DNA replication forks

“…RPA1 is one of the most important eukaryotic single-strand DNA-binding proteins, and serves to protect stalled DNA replication forks. It is also a critical factor in DNA metabolism [11,12,34,35]. Identification of the functional linkage between PTEN and RPA1 in this study therefore raises the possibility that PTEN is involved in other as yet unidentified aspects of DNA biology.…”

Single strand DNA binding proteins 1 and 2 protect newly replicated telomeres

“…During S-phase, POT1a/b is displaced transiently from telomeres by RPA, possibly as a result of the arrival of the replication machinery at telomeres [32,33]. The subsequent RPA to POT1a/b switch on telomeres in turn depends on additional factors, including telomere repeatcontaining RNA and heterogeneous nuclear ribonucleoproteins A1 and A2/B1 [32]. The discovery that mSSB1 and mSSB2 are also required to protect newly replicated telomeres adds additional complexities to this scenario.…”

“…After DNA replication of lagging-strand telomeres, POT1a/b binds to and protects the 3′ overhang, while at leadingstrand telomeres 5′ to 3′ nucleolytic processing by Apollo generates the 3′ overhang for POT1a/b to load on [15,25]. During S-phase, POT1a/b is displaced transiently from telomeres by RPA, possibly as a result of the arrival of the replication machinery at telomeres [32,33]. The subsequent RPA to POT1a/b switch on telomeres in turn depends on additional factors, including telomere repeatcontaining RNA and heterogeneous nuclear ribonucleoproteins A1 and A2/B1 [32].…”

“…A model has emerged on how ssDNA-binding proteins POT1a/b and RPA coordinate telomere end protection and DNA replication, respectively [13,32]. After DNA replication of lagging-strand telomeres, POT1a/b binds to and protects the 3′ overhang, while at leadingstrand telomeres 5′ to 3′ nucleolytic processing by Apollo generates the 3′ overhang for POT1a/b to load on [15,25].…”

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

“…The failure to observe immediate ATR activation and RPA binding to telomeres suggests that either the amount of ss DNA was insufficient to activate ATR, or RPA was precluded from binding to the accumulated ss DNA in Stn1-deficient cells. This is perhaps due to the presence of Pot1 (see below, Figure 6), which suppresses ATR activation at telomeres by preventing RPA binding to telomeres [35][36][37][38][39].…”