Mechanism of the Human Telomere C-Strand Fill-In Machinery Assembly and Activation at Chromosome Ends

Project Summary/Abstract Telomeres are specialized nucleoprotein structures that protect chromosome ends and confer genome stability in eukaryotic cells. Without telomeres, chromosomes undergo misguided end-to-end fusions, leading to chromosomal aberrations and eventual genome instability. Hence, deregulation of telomere maintenance results in human diseases such as cancer and dyskeratosis congenita. The human telomeric DNA comprises thousands of tandem repeats of the conserved hexameric sequence, TTAGGG. The telomeric DNA has a unique structure, a several kilobases long double-stranded DNA (dsDNA) region that ends with a 3' single- stranded DNA (ssDNA) overhang. The cornerstone of telomere maintenance is the two-part process of telomeric DNA synthesis. In the first step, the ribonucleoprotein telomerase extends the telomere G-rich 3' overhang. Telomerase adds telomeric repeats to the ssDNA overhang. Next, telomeric ssDNA-binding protein complex, CTC1-STN1-TEN1 (CST), coordinates with DNA polymerase alpha-primase (Polα-primase) to fill in the newly-synthesized telomeric G-overhang by de novo C-strand synthesis. While the mechanism of the extension of telomeric G-overhang by telomerase is an area of intense study since the discovery of telomerase almost four decades ago, that for the telomeric C-strand fill-in by CST-polα-primase is much less understood. Telomere C-strand fill-in is equally essential in telomere maintenance as the G-overhang extension process. As such, this proposal aims to study the molecular mechanism of the human telomere C-strand fill-in machinery by providing biochemical and structure-function relationship understandings of template-bound CST-Polα- primase at key catalytic steps. Multiple novel models and hypotheses from this proposal are based on the premises of our recent cryogenic-electron microscopy (cryo-EM) structures of the human template-bound CST- Polα-primase preinitiation complex (PIC). For the first aim, we will establish an unprecedented consensus template sequence for CST-Polα-primase assembly and C-strand synthesis initiation. For the second aim, we will use cryo-EM single-particle analysis to determine the structural basis of how CST-Polα-primase initiates de novo RNA primer synthesis after PIC assembly at telomeric overhangs. For the third aim, we will elucidate the molecular mechanism of how CST-Polα-primase “counts” the RNA primer length during synthesis and promptly terminate the “matured” RNA primer for the intramolecular primer handover to the DNA polymerase domain. The findings from this proposed work will provide a timely advancement to our understanding of the human telomere C-strand fill-in mechanism and mammalian telomeric DNA synthesis in general. The proposal structure-function studies provide a missing platform to connect CST and Polα-primase human disease mutations to mechanistic understandings. From a broader perspective, telomere C-strand fill-in is an excellent model for studying lagging-strand synthesis by Polα-primase in replisomes.