TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

Pan, H.; Kaur, P.; Liu, M.; Xu, P.; Mahn, C.; Barnes, R.; Tang, Q.; Hao, P.; Bhattaram, D.; You, C.; Piehler, J.; Weninger, K.; Riehn, R.; Smith, S.; Opersko, P. L.; Wang, H.

doi:10.1101/2020.09.12.286559

bioRxiv · 10.1101/2020.09.12.286559

TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

Pan, H.·Kaur, P.·Liu, M.·Xu, P.·Mahn, C.·Barnes, R.·Tang, Q.·Hao, P.·Bhattaram, D.·You, C.·Piehler, J.·Weninger, K.·Riehn, R.·Smith, S.·Opersko, P. L.·Wang, H.

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Abstract

The shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3 overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

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Pan, H., Kaur, P., Liu, M., Xu, P., Mahn, C., Barnes, R., Tang, Q., Hao, P., Bhattaram, D., You, C., Piehler, J., Weninger, K., Riehn, R., Smith, S., Opersko, P. L., Wang, H.. 2020-09-13. TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA. https://doi.org/10.1101/2020.09.12.286559

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