Abstract
Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.
Highlights
Telomeres protect chromosome ends from degradation, DNA rearrangements, and DNA damage signaling, which are seen at DNA double-strand breaks (de Lange 2009; Denchi and Sfeir 2016)
We depleted TRF1 and TRF2 to assess their roles in telomere compaction and used a large field of view (FOV) flat illumination microscope setup to capture a large number of telomeres (>900 per condition) with high image quality (Douglass et al 2016)
To visualize the TTAGGG repeats of human telomeres, we hybridized fixed HeLa cells with a PNA oligonucleotide (5′-CCCTAA-3′)3 probe that was labeled at its 5′ end with the fluorescent dye Alexa fluor 647
Summary
Telomeres protect chromosome ends from degradation, DNA rearrangements, and DNA damage signaling, which are seen at DNA double-strand breaks (de Lange 2009; Denchi and Sfeir 2016). Stalled replication forks accumulate ssDNA, which, when bound by replication protein A, recruits ATRIP–ATR to initiate a DNA damage response (DDR) (Zou and Elledge 2003) This can explain how TRF1-depleted telomeres activate the ATR checkpoint kinase in S phase. A very different alternative model was proposed recently in which loss of TRF2 would lead to an up to 10-fold decompaction (decrease in density) of telomeric chromatin, rendering telomeres accessible to DDR factors that would otherwise be excluded (Bandaria et al 2016) Within this model, activation of ATR signaling upon TRF1 depletion was explained by chromatin decompaction rather than the accumulation of ssDNA at stalled replication forks in S phase as discussed above. Our results reveal that the vast majority of DDR-positive telomeres does not differ in size from DDR-negative telomeres, excluding telomere decompaction from being generally associated with the DDR
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