Determination of telomeric protein interaction binding sites between amino acids 58-268 of TRF1 and 224-276 of TIN2 and between amino acids 42-245 of TRF2 and 203-361 of TIN2

Abstract

Interactions of telomeric protein-protein complexes have important implications in the development of diseases such as cancer. Approximately 90% of all cancer cases are associated with the reactivation of telomerase (Cech, 2004). Therefore, it is imperative to determine the protein interactions involved in the down-regulation of telomerase. This study focused on two central telomeric proteins, TRF1 and TRF2, and their interaction with another telomeric protein, TIN2. These proteins are involved with the T -loop formation, which helps protect chromosome ends from damage. Previous experiments have shown that the alteration in the binding of TRF I, TRF2, or TIN2 may cause either cell apoptosis or even uninhibited cell replication (Liu et aI., 2004, Zheng-Sheng Ye et ai., 2004). In order to study the interactions, gel filtrations columns were used to detect the formation of protein-protein complexes in solutions. Smaller fragments were then designed in order to determine the site of interaction to a more specific sequence of nucleotides. As a result of this experiment, the nucleotide sequence between TRF 1 and TIN2 was narrowed to amino acids 58-268 and 224-276, respectively. The interaction between TRF2 and TIN2 was then narrowed to a small number of amino acids, 42-245 and 203-361, respectively. This research also demonstrated competitive binding between TRF 1 and TRF2 to TIN2. When a complex ofTRF2!fIN2 was introduced to the protein TRFI, TRFI bound preferentially to TIN2, releasing TRF2. This contradicts earlier studies that both TRFI and TRF2 bound simultaneously. Other telomeric proteins should be examined for competitive binding, in order to fully understand the workings of the telomeric complex. Telomere research is important because an in-depth knowledge of the complex interactions could allow for the development of drugs that target those interactions and therefore protect the proteins from interference.