Abstract
The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3' ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.
Highlights
HTRF2 and hRap1 prevent non-homologous end joining and exist as a complex at human telomeres
Short DNAs do not recapitulate the full telomere architecture, and we used EM to examine the DNA binding ability of hRap1 using DNA templates in the range of 0.7 to 7 kb, including the minichromosomes, Holliday Junctions and replication forks illustrated in Fig. 1, A–C
To understand the role of hRap1 at telomeres, we studied the DNA binding characteristics of hRap1 and the TRF2-Rap1 complex on different DNA templates that mimic telomeric structures
Summary
Preparation of Linear Telomeric Templates—The pRST5 plasmid contains ϳ575 bp of ds human telomeric DNA [9]. To generate a template with a telomeric 5Ј overhang, BsmBI digested pOST6 was treated with ExoIII on ice for 5 min per the manufacturer’s instructions (New England Biolabs). A nontelomeric template with a 3Ј overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats [29], with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs). To generate a radiolabeled telomeric template with a 3Ј overhang, the gel isolated duplex DNA was ligated to a G-rich oligonucleotide with eight telomeric repeats after the oligonucleotide was phosphorylated with [␥-32P]ATP from its 5Ј end as described above. NH2-terminal His6-tagged hTIN2 was purified with nickel-nitrilotriacetic acid chromatography (Qiagen) from Sf21 extracts [33] and stored at Ϫ80 °C in 50 mM NaPO4, 150 mM NaCl, 8 mM -mercaptoethanol, and 20% glycerol. To determine the elution profile of the individual components, 100 g of NH2-terminal His6-tagged hTRF2 or NH2-terminal His6-tagged hRap proteins were passed through the Sepharose 6 size exclusion column (GE Healthcare) in a buffer of 300 mM NaCl, 20 mM Hepes (pH 8.75), and 8 mM -mercaptoethanol
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