Mechanisms regulating telomere length, protection and replication

PROJECT SUMMARY Telomeres are TTAGGG repetitive DNA-protein complexes at chromosome ends required to solve three significant problems critical for the maintenance of genome stability. The “end replication problem” is due to the inability of DNA polymerase to completely copy the lagging strand of chromosome ends, necessitating telomerase to synthesize the 3’ G-strand. The CTC1-STN1-TEN1 complex recruits DNA primase-polymerase alpha to promote fill-in synthesis of the 5’ C-strand. A major gap in our knowledge is how G- and C-strand synthesis are coordinated to maintain telomere length. We have previously shown that the POT1 protein participates in both G- and C-strand synthesis at mouse telomeres. To determine whether human POT1 coordinates telomere length maintenance, we will use state-of-the-art biochemical, structural, cellular and genetic approaches to explore the possibility that a phosoho de-phopho switch occurs on hPOT1 to coordinate the recruitment of telomerase and CST to telomeres to maintain telomere length. The telomere “end protection problem” is due to the fact that telomere ends resemble DNA double-strand breaks, which must be protected by the shelterin complex to prevent the activation of a DNA damage response. We have previously shown that shelterin components TRF2-RAP1 represses telomere homology directed repair (HDR). To determine mechanistically how TRF2-RAP1 protects telomeres from engaging in HDR, we conducted genome-wide CRISPR screens and IP-mass spectrometry. We identified BLM and ADAR1p110 as potential candidates proteins involved in telomere HDR. Using purified proteins, we developed a novel telomere D-loop strand invasion assay and a telomere D-loop unwinding assay to address how BLM participates in telomere HDR. We also developed a telomere R-loop assay to determine mechanistically how telomere R-loops are resolved by ADAR1p110. The “end replication problem” stems from the observation that telomeres are difficult to replicate regions in the genome. To address the gap in knowledge of what novel proteins are involved in telomere replication, we used BioID to identify Claspin as a TRF2 interacting protein involved in DNA replication. We will determine mechanistically how Claspin resolves telomere replication stress, using a telomere single-molecule analysis of replicated DNA to measure the speed of telomere replication fork progression. Using this system, we will also test whether TRF2 binding proteins identified in our CRISPR screens are required for telomere replication. We invented an innovative technique called COMET-FISH that combines strand-specific chromosome-orientation (CO)-FISH with immuno-metaphase-FISH, to visualize C- or G-strand-specific localization of Claspin to telomeres during replication stress. Our proposed research will yield new mechanistic insights into how telomeres are protected, maintained and replicated, with broad future implications for understanding the treatment of human diseases including cancer and aging.