TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA - PubMed (original) (raw)

. 2011 Mar 24;471(7339):532-6.

doi: 10.1038/nature09772. Epub 2011 Mar 13.

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

Rachel Litman Flynn et al. Nature. 2011.

Abstract

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 telomeres, 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 telomeres. 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.

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Figures

Figure 1

Figure 1. A novel telomere-specific RPA displacing activity in human cell extracts

a, POT-TPP1 (60 nM; purified from insect cells), RPA (60 nM), and mixtures of POT1-TPP1 and RPA (60, 120, 180 nM of POT1-TPP1 mixed with 60 nM of RPA) were incubated with 20 nM of the ssDNA probe and analyzed by gel-shift. b, POT1-TPP1 (2.4 nM), RPA (2.4 nM), and mixtures of POT1-TPP1 and RPA (2.4, 4.8, 7.2 nM of POT1-TPP1 mixed with 2.4 nM of RPA) were incubated with 0.8 nM of biotinylated ssTEL [(TTAGGG)8]. The proteins bound to ssTEL were retrieved by streptavidin beads and analyzed by Western blot. c, Biotinylated ssTEL or ssMUT [(TTTGCG)8] was incubated with WCEs or recombinant RPA (rRPA). d, ssTEL or ssMUT precoated with RPA was incubated with increasing concentrations of HeLa WCEs (0.08, 0.19, 0.36, 0.8, 1.3 μg/μl). The RPA2 remaining on ssTEL was analyzed as in b. e, ssTEL precoated with POT1 was incubated with increasing concentrations of HeLa WCEs (0.07, 0.18, 0.33, 0.66, 1.3 μg/μl).

Figure 2

Figure 2. RPA displacement by hnRNPA1

a, ssTEL and ssMUT precoated with RPA were incubated with NEs. After the incubation, the proteins bound to DNA were retrieved, eluted, and applied to RPA-coated ssTEL or ssMUT (see Supplemental Methods). After the second incubation, the remaining RPA2 on DNA was analyzed by Western blot. b, Proteins captured by RPA-ssTEL or RPA-ssMUT and eluted by salt were analyzed by Western blot using antibodies to hnRNPA1, hnRNPA2/B1, and TRF2. c, RPA-coated ssTEL (0.8 nM) was incubated with increasing concentrations of WCEs (0.06, 0.24, 0.96 μg/μl). The hnRNPA1 and hnRNPA2/B1 bound to DNA and the remaining RPA2 on DNA were analyzed by Western blot. d, RPA-coated ssTEL or ssMUT (0.8 nM) was incubated with increasing concentrations of purified hnRNPA1 (2.4, 4.8, 7.2 nM). The remaining RPA2 on DNA was analyzed as in a. e, POT1-coated ssTEL (0.8 nM) was incubated with increasing concentrations of purified hnRNPA1 (2.4, 4.8, 7.2 nM).

Figure 3

Figure 3. Regulation of RPA displacement by TERRA

a, NEs (34 ng/μl) were treated with increasing concentrations (2, 4, 10, 20 nM) of TERRA (UUAGGG)3, control RNA (CCCUAA)3, or mock treated. The treated NEs were then incubated with RPA-coated ssTEL (2 nM), and the remaining RPA2 on ssTEL was analyzed after the incubation. b, The RPA displacing factors were captured with RPA-ssTEL as in Fig. 2a. The elution was incubated with TERRA or control RNA, and then applied to RPA-ssTEL. c, Purified hnRNPA1 (4.8 nM) was incubated with increasing concentrations of TERRA or control RNA (2, 4, 10, 20 nM), and then incubated with RPA-ssTEL (0.8 nM). d, hnRNPA1-coated ssTEL (0.8 nM) was incubated with increasing concentrations of TERRA (2, 20, 200, 2000 nM). The remaining hnRNPA1 on ssTEL was analyzed by Western blot. e, hnRNPA1-coated ssTEL (0.8 nM) was incubated with increasing concentrations of TERRA (2, 20, 200 nM) in the presence of POT1-TPP1 (2.4 nM). The hnRNPA1 and POT1 on ssTEL were analyzed by Western blot. f, A model for RPA displacement.

Figure 4

Figure 4. hnRNPA1 and POT1 suppress the accumulation of RPA at telomeres

a, RPA-coated ssTEL was incubated with WCEs from cells in G1, early S, late S, and M phases of the cell cycle (see Supplemental Methods). The remaining RPA2 on ssTEL was analyzed after incubation. Cyclin A and phospho-histone H3 serve as cell-cycle markers, and histone H4 as a loading control. b, TERRA was analyzed by RNA FISH in HeLa cells following thymidine release. TRF2 severs as a marker of telomeres. c, HeLa cells were treated with hnRNPA1 siRNA or mock treated, and then immunostained with antibodies to RPA2 and TRF2 (left panel). The cells with RPA foci (>5) were quantified (right panel). Mean+s.d., n=3 for mock and sihnRNPA1-1, n=2 for sihnRNPA1-2. d, Chromatin immunoprecipitation of RPA was performed with two different RPA2 antibodies. The association of RPA with telomeres was analyzed by dot blot using a telomere probe and quantified. e, HeLa cells transfected with POT1 or LacZ siRNA were released from a thymidine block. At the indicated times, the G2/M population was determined by FACS (see Fig. S10c). Cells were immunostained for RPA2 and TRF2 (left panel). The cells with RPA foci (>5) were quantified (right panel). Mean+s.d., n=3.

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References

    1. Guo X, et al. Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis. Embo J. 2007;26:4709–19. - PMC - PubMed
    1. Denchi EL, de Lange T. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature. 2007;448:1068–71. - PubMed
    1. Porro A, Feuerhahn S, Reichenbach P, Lingner J. Molecular dissection of TERRA biogenesis unveils the presence of distinct and multiple regulatory pathways. Mol Cell Biol - PMC - PubMed
    1. Wold MS. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem. 1997;66:61–92. - PubMed
    1. Baumann P, Cech TR. Pot1, the putative telomere end-binding protein in fission yeast and humans. Science. 2001;292:1171–5. - PubMed

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