T-loops Refold after Telomerase Extension and Unfold Again at Late S/G2 for C-strand Fill-in

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 D-loop. It is unknown whether this D-loop is the size of the overhang, or whether branch migration results in a larger D-loop. 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. HighlightsO_LIT-loops unfold twice during each S-phase C_LIO_LIVirtually all telomeres are packaged into t-loops C_LIO_LILarge amounts of t-loops can easily be purified without specialized materials C_LI