Defining Multiple Steps in Human Telomere End Processing
Telomere overhangs are essential for chromosome end protection and telomerase extension, but how telomere overhangs are generated is unknown. Due to the classic end replication problem, leading DNA daughter strands are initially blunt while lagging daughters are shorter by at least the size of the final RNA primer, which historically is believed to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. Understanding the number and nature of the overhang processing events is crucial in establishing the roles of candidate proteins involved. We here define these steps in normal human cells. We show the final lagging RNA primer is positioned ~70-100 nt from chromosome ends (not at the extreme ends), and is not removed for ~1hr following replication. Therefore, the location of the RNA primer, rather than its size, is a primary driving force for telomere shortening. Moreover, we demonstrate that telomere end-processing occurs in two distinct phases following telomere duplex replication. During the early phase, which occupies 1-2 hours following telomere replication, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2 when the C-terminal nucleotide is specified, referred to as the late phase. Furthermore, in human cancer cells under maintenance condition, telomerase extension is uncoupled from C-strand fill-in. These results uncover crucial mechanistic details of the DNA end-replication problem as well as several specific steps in telomere overhang processing. These results also indicate the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes. The findings and the methods developed will now provide the basis for examining candidate factors that may function to regulate particular steps in telomere length homeostasis with implications in both cellular aging and cancer.
Biochemical Processes, Telomere Shortening, DNA Replication
University of Texas Southwestern Medical Center
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