Telomere overhangs are essential for telomere end protection and telomerase extension, but how telomere overhangs are generated is unknown. Leading daughter strands synthesized by conventional semiconservation DNA replication are initially blunt, while lagging daughter strands are shorter by at least the size of the final RNA primer, which is thought to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. We show that the final lagging RNA primer is not terminal but is randomly positioned~70-100 nucleotides from the ends and is not removed for more than an hour. This identifies an important intrinsic step in replicative aging. Telomeric termini are processed in two distinct phases. During the early phase, which occupies 1-2 h following replication of the duplex telomeric DNA, 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. These observations suggest the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes.
Telomeres are the structures that protect the ends of each chromosome and prevent them from being recognized as broken DNA in need of repair. During human fetal development telomere length is ~15 kb and is maintained by the ribonucleoprotein telomerase. In neonates telomerase becomes spliced into inactive forms in most tissues. Afterwards, lagging strand synthesis fails to copy the very end and telomeres become progressively shorter as cells divide. Part of the mechanism for avoiding the DNA damage machinery is by forming a t-loop structure that has no free end. The telomeric 3' overhang is inserted into the duplex DNA where it displaces the G-rich strand, forming a Dloop. It is unknown whether this D-loop is the size of the overhang, or whether branch migration results in a larger Dloop. Telomere t-loops must unfold for replication and telomerase extension during S phase. It is also unknown whether t-loops remain unfolded after replication until C-strand fill-in at late S/G2, or if they refold after replication and then unfold again for fill-in at late S/G2. We have developed a biochemical t-loop assay to address these issues, and demonstrate that most t-loops persist throughout S phase both before and after their replication.
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