Project Summary/Abstract Our laboratory is interested in understanding how eukaryotic cells ensure the maintenance of telomeres, the natural ends of linear eukaryotic chromosomes. Evolutionarily conserved shelterin and CST (CTC1/Cdc13- STN1-TEN1) complexes play essential roles in telomerase recruitment and protection of telomeres against DNA repair and checkpoint factors. Stable maintenance of telomeres is critical to preserve genomic integrity and prevent accumulation of undesired mutations that might lead to tumor formation. Regulation of telomere structures and telomerase also affect cell proliferation and tissue maintenance in aging organisms. Therefore, basic mechanistic studies investigating how telomere and DNA damage response proteins collaborate in proper telomere maintenance should provide critical insights necessary to help devise more effective treatment strategies against tumors or other age-related diseases. Our proposed research projects utilize fission yeast Schizosaccharomyces pombe. Fission yeast telomeres serve as a good model for human telomeres, since proteins involved in telomere maintenance are highly conserved between fission yeast and humans. Studies from our lab and others have provided detailed insights how fission yeast shelterin and Stn1-Ten1 ensure stable maintenance of telomeres in fission yeast. Those include findings that (1) Tel1ATM/Rad3ATR_ dependent phosphorylation of the shelterin subunit Ccq1 on Thr93 promotes telomerase recruitment by promoting interaction between Ccq1 and the telomerase subunit Est1, and (2) SUMOylation of another shelterin subunit Tpz1ree, on Lys242 facilitates Stn1-Ten1 recruitment to telomeres and limits telomere extension. Evolutionarily conserved "TEL patch" residues within Tpz1 have also been found to promote telomerase activation and recruitment, further highlighting the well conserved nature of telomere regulation by fission yeast and mammalian shelterin. Our analyses of temporal binding patterns for DNA polymerases, telomerase, shelterin and Stn1 found that shelterin subunits Rap1 and Poz1 and the Stn1-Ten1 complex promote timely dissociation of telomerase from telomeres by promoting recruitment of Pola to complete lagging strand synthesis at telomeres. For the current grant application, our proposed experiments will (1) identify and characterize underlying regulatory mechanism(s) that allow Ccq1 and Poz1 to promote Pola-dependent telomere protection (Aim 1), (2) identify new interaction partners of Stn1-Ten1 complex and characterize their contributions to recruitment/retention of Stn1-Ten1 complex at telomeres and non-telomeric sites (Aim 2), and (3) investigate how regulation of TERRA vs. poly(A)+ TERRA expression modulates Stn1-Ten1-Pola recruitment at telomeres (Aim 3).