Examination of different APIM-variants and APIM's role in TRF2 protection of the telomere ends
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Most chemotherapeutic drugs induce DNA damage to the cells. However, the toxicity of DNA damaging drugs can be reduced by DNA repair mechanisms, and the cancer cells can survive despite the damage. Therefore, by targeting proteins involved in DNA repair the efficacy of chemotherapeutic drugs can be increased. Proliferating cell nuclear antigen (PCNA) is essential in a variety of processes involved in DNA replication, cell cycle control and DNA repair, where it functions as a scaffold protein that orchestrates all the events played out by the PCNA binding proteins. Numerous proteins that interact with PCNA are thought to do so via conserved PCNA binding motifs, either the PCNA-interacting protein (PIP)-box or the newly discovered AlkB Homologue 2 PCNA-interacting Motif (APIM). The APIM-containing proteins are involved in epigenetics, genome maintenance, and cell cycle control and many of the proteins are important after DNA damage. Overexpression of APIM-peptides is found to increase cancer cells´ sensitivity to chemotherapeutic drugs. Telomeric repeat binding factor 2 (TRF2) is an APIM-containing shelterin protein that represses the Ataxia Telangiectasia Mutated (ATM) kinase pathway at telomeres. Loss of TRF2 will lead to activation of this pathway and telomeres will be recognized as sites of DNA damage. The consequence can be seen as telomere dysfunction induced foci (TIF) at chromosome ends, which are telomere ends associated with DNA repair factors such as γ-H2AX. Overexpression of TRF2 is found to protect telomeres from the damage repair machinery during mitotic arrest. The aims of this master thesis have been to examine new APIM-variants´ ability to bind to PCNA, in vivo, using confocal imaging and fluorescence resonance energy transfer (FRET), to examine the different APIM-variants´ ability to sensitize cells to cisplatin. Furthermore to investigate if APIM in TRF2 was important for its role in protecting telomere ends. In total eight new APIM-variants were proven functional. A likely new APIM consensus would be (K/R)-(F/Y/W)-(L/I/V/A/F)-(L/I/V/A/G/S)-(I/V)-(R/K). The red letter indicates amino acids removed from the earlier suggested consensus, while the green indicates new possible amino acids. New APIM-variantswere found to be able tosensitize HeLa cells to cisplatinto a certain degree, however, noneof the variants tested were found to sensitize the cells strongerthan APIM WT. Results indicatedthat APIMin TRF2 is a functional PCNA binding motif as mutatedAPIM impairedTRF2s role in protecting the telomeres via a PCNA dependent process.