Title: TIN2 is and Architectural Protein Stabilizing TRF1 at Telomer DNA

Abstract: Telomeres, consisting of duplex TTAGGG repeats and associating with protein complexes at chromosome ends, play a crucial role in maintaining the stability of chromosomes. The protein complex – shelterin contains six subunits (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1), which bind to telomeres and protect the chromosome ends from DNA repairing, and recruit telomerase when chromosome gets shortened abnormally. Among these six subunits, TIN2 has no affinity to either double-strand or single-strand DNA. However, it is a core component bridging the double-strand DNA binding proteins (TRF1 and TRF2) to single-strand DNA binding protein complex (TPP1-POT1). Loss of TRF1 or TRF2 binding domain in TIN2 can trigger DNA damage response. Moreover, TIN2 without TPP1 binding domain is capable of fully supporting the stabilization of TRF1 and TRF2/RAP1. Despite the significance of TIN2 in telomere maintenance, the mechanism underlying TIN2 remains elusive. To understand how TIN2 affects TRF1 binding dynamics and how TIN2 influences with TRF1-mediated telomeric DNA pairing, we conducted Atomic Force Microscopy (AFM) and used nanochannels confined DNA to study the telomeric DNA conformation upon TRF1 and TIN2 binding. We observed high-order protein-DNA complexes formation indicating TIN2 facilitates TRF1 accumulation on telomeric DNA. We also carried out single molecule fluorescence technique to investigate how TIN2 affects TRF1 binding dynamics on telomeric DNA sequences. Our results show that TIN2 can accelerate DNA-DNA pairing and stabilize TRF1 on telomeric DNA