Dna Base Damage And Consequential Point Mutation Controls Telomere Conformation And Elaborates Telomerase Extension Activity

Telomeres are protein-DNA complexes that cap the ends of chromosomes and protect the chromosomes from deterioration. Stem cells and most cancer cells maintain telomere length by activating telomerase, a reverse transcriptase that extends telomeric overhang by adding TTAGGG tandem repeats. Telomere is considered a hot spot for oxidative damage due to its high guanine content. We used single-molecule FRET (smFRET) microscopy to test if different positions and types of oxidative damages impart unique effect in telomere structure and telomerase activity. Our recent work showed that the G-quadruplex (GQ) formed by telomeric overhang is destabilized by a single site damage, leading to an increased telomerase activity. Here, we compared two common oxidatively damaged bases, 8oxo-guanine and O6-methylguanine, in three terminal guanine positions of telomere, which is the key access point for telomerase. Consistent with previous findings, any modification of the second position is most disruptive while the other two positions are better tolerated. At all positions, the structural instability induced by oxidative damage, especially the O6-methylguanine, far exceeds the perturbation by base substitutions, rendering high accessibility to complementary DNA strand. Telomerase binding and extension activity is diminished greatly by the two consequential point mutations (T and A) at the terminal guanines regardless of the position or structural disruption, likely due to the base-paring dependent mode of binding. In contrast, point mutations in upstream guanines causes same level of telomerase binding as oxidatively damaged bases at the terminal guanines. Our finding demonstrates how a single base damage can drive the structural alteration and lengthening of telomere in a position dependent manner. Furthermore, this data suggests a long-term and inheritable effect of oxidative damage on telomere lengthening.