Examinations of the functionality of APIM in TRF2 and the importance for telomere stability
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Maintaining genome integrity is crucial for life, and therefore, complex control and repair mechanisms have evolved. Because cells are especially susceptible to damage during replication, many DNA repair pathways are coupled to the replication machinery. Proliferating cell nuclear antigen (PCNA) is a scaffold protein that bind many of the proteins involved in these processes, and is important in orchestrating replicative and repair events. PCNA’s binding partners bind to it via two conserved binding motifs, the PCNA interacting peptide (PIP)-box and AlkB homologue 2 PCNA-interacting motif (APIM). Because PCNA regulates many different replication and repair pathways, PCNA-protein interaction is a potent cancer therapy target, as the inhibition of PCNA interaction is less likely to be circumvented by alternative pathways. Many APIM containing proteins are associated with DNA repair, and the blocking of APIM has been shown to sensitize cells to cytotoxic agents. Cancer cells, which have acquired unlimited proliferative potential, are dependent on continuous lengthening and maintaining of telomeres. Telomerase and the telomeres themselves, are therefore being explored as targets for cancer therapy. The shelterin protein TRF2 is important for maintaining the telomeric t-loop, which protects the telomeres from being recognized by the DNA damage repair system as double stranded breaks (DSBs). The dissociation of TRF2 from telomeres following prolonged mitotic arrest, leads to telomeric deprotection, accumulation of DNA damage associated factors at telomeres, and activation of damage response pathways, visualized as telomere damage-induced foci (TIFs). TRF2 is known to have APIM, but the motif is not proven to be functional. We wanted to examine the functionality of APIM in TRF2, and investigate its importance, if any, for telomere protection. We studied this by investigating the influence of intact APIM in TRF2 on the formation of TIFs after colcemid-induced mitotic arrest. We also examined if the interaction of APIM-containing proteins with PCNA is important for cells’ ability to recover from mitotic arrest, by using the APIM containing peptide ATX-101. We also asked if APIM in TRF2 has a role in replication and repair of telomeric sequences after UV damage. To examine this, we used SupF mutation reporter vectors with telomeric repeats of two different lengths upstream in the SupF gene, which makes it possible to study TRF2’s role in replication and repair of telomeric sequences independently of its role in stabilizing the telomeric t-loop structure. Our results indicate that APIM in TRF2 is a functional binding motif, and suggests that the regulation of TRF2 activities probably is linked to PCNA interaction via APIM. Furthermore, ATX-101 was found to increase the cytotoxic effect of colcemid some degree, suggesting that interaction of APIM-containing proteins with PCNA has a role in cells’ ability to survive metaphase arrest. APIM in TRF2 also seems to have a role in repair of telomeric sequences after UV-damage, and this TRF2-activity is therefore probably regulated by TRF2-PCNA binding via APIM.