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Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.
Figure 1. Generation and characterization of telomeric DNA substrates. (A) pRST5 contains an insert of 98 telomeric repeats (bold), which is excised by BsmBI/HindIII to generate 0.6 kb linear substrates containing exclusively telomeric DNA. Overhangs are filled by T4 DNA polymerase, dA/G/TTP and biotinylated dCTP. After incubation of immobilized substrates for 20 min in cytosolic extracts, proteins in the soluble fraction or bound to DNA are monitored by western blotting. Non-biotinylated DNA is used for replication and NHEJ assays. A NT control plasmid pRST5_NT harbors a random sequence of similar length. (B) Bead-bound NT or telomeric (T) DNA was incubated with cytosolic extracts at 2 à 109 ends/µl. Soluble fractions were separated (lanes 1â3), beads washed and bead-bound fractions (lanes 4â6) analyzed by western blotting. Lanes 3 and 6, beads only.
Figure 2. Assembly of pre-RC components on telomeric DNA. (A) Assembly of pre-RC proteins on immobilized 0.6 kb telomeric or NT DNA in cytosolic extracts as described in Figure 1B. A part of the membrane stained with Ponceau was used as an internal loading control. (B) Immobilized 0.6 kb NT or telomeric substrates were incubated with egg cytosol at 30 ng/µl for 30 min before addition of 2-fold volumes of NPE. Reactions were processed after 25 min as in Figure 1B. 100 ng/µl Geminin was added to the egg cytosol in lanes 7 and 8. (C) Quantification of the signals for TRF2, ORC2 and MCM6 from (B) for three independent experiments. Signals were normalized to the signal of the Ponceau stain. Values for untreated NT conditions are set to 1.
Figure 3. Replication of linear substrates. (A) BsmBI linearized 3.5 kb NT pRST5_NT (5_NT) was incubated with egg cytosol at 1.5 ng/µl in the presence of either buffer control, 5 mM Caffeine, 100 ng/µl Geminin or Caffeine and Geminin for 30 min before addition of 2-fold volumes of NPE and (α-32P)dCTP. Reactions were processed after 60 min for native agarose gel electrophoresis. The radioactive signal of three independent experiments were quantified by Phosphorimager (B). Values for untreated conditions are set to 1. P-values were calculated using a t-test.
Figure 4. Telomeric DNA is replicated. (A) Replication assays with 3 ng/µl of 0.6 kb NT (lanes 1â4) or telomeric (lanes 5â8) DNA were performed as in Figure 3A. Reactions were processed after 40 min. (B) Quantification of replication assays from three (NT) or 4 (T) independent experiments. Values for untreated conditions are set to 1. P-values were calculated using a t-test from Caffeine versus Geminin treated extracts, as indicated by asterisks.
Figure 5. Short telomeric substrates activate a DSB response and are repaired by NHEJ. (A) Southern blot analysis of NHEJ-mediated repair products. In total, 0.6 kb of untagged NT or telomeric DNA was added to cytosolic extracts for 150 min. Reactions were processed for native agarose gel electrophoresis before transfer onto a Nitrocellulose membrane. Telomeric substrates are hybridized with a 5Ⲡ32P-labeled (CCCTAA)6 oligonucleotide and NT substrates with random labeled pRST5_NT. LM linear monomer, LD linear dimer, M multimers. Signals from three independent experiments were quantified by phosphorimager analysis and expressed as ratios of the LD to LM. (B) Immobilized NT or telomeric DNA was incubated with cytosolic extracts at indicated concentrations and activation of DNA damage response proteins in the soluble fraction was monitored by western blotting. Lane 9, buffer control, lane 10, 2 ng/µl HaeII-digested pBS was added. (C) Immobilized NT or telomeric DNA was incubated with cytosolic extracts at 2 à 109 DSB/µl and checkpoint activation in the soluble fraction was monitored by western blotting using an antibody against phosphorylated Chk1. Beads only, lane 5, 2 ng/µl HaeII digested pBS, lane 6, buffer control, lane 7.
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