Telomere loops regulate activation of the DNA damage response by the chromosome end — ASN Events
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  3. Telomere loops regulate activation of the DNA damage response by the chromosome end

Telomere loops regulate activation of the DNA damage response by the chromosome end (#259)

David Van Ly 1 , Sonja Frölich 2 , Ronnie Ren Jie Low 1 , Katharina Gaus 3 , Tony Cesare 1
  1. Genome Integrity Group, Children's Medical Research Institute, Westmead, New South Wales, Australia
  2. Robinson Research Institute, University of Adelaide, Adelaide, South Australia, Australia
  3. EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, New South Wales, Australia

Telomere deprotection, in humans, progresses through an “intermediate-state”, which is receptive to DNA damage response (DDR) activation, but suppressive of telomere-telomere fusion. We previously proposed that DDR activation at telomeres is suppressed by t-loop formation. Our hypothesis was, that failure to adopt a t-loop exposed the chromosome terminus as an ATM-activating substrate, while DNA repair activity remained suppressed at the chromosome end through residual TRF2 binding to the telomeric DNA. Telomere fusions then only occur after TRF2 is almost completely removed from the chromosome end. The development of super-resolution microscopy methods to visualize t-loops, has enabled us to test this predication.

Here we probed telomere structure in mammalian cells using STORM, STED, SIM and Airyscan super-resolution microscopy. We developed a temporally controlled system using TRF2F/- Cre-ER LgT MEF cells, where following gene deletion, the telomeres reproducibly transition through the three-states of telomere protection. Super-resolution imaging revealed that the percentage of looped telomere molecules decreased as a function of DDR-activation, before telomere fusions occurred. However, the length distribution of the telomeric DNA in the looped (loop + tail) and linear forms, did not change during this transition, consistent with telomere DDR activation corresponding with loss of t-loop structure. Human HeLa LT cells, transduced with TRF2 shRNAs of different efficiencies, revealed that looped telomere molecules were only reduced concomitant with the induction of a telomere DDR, but not in cells where the shRNA reduced TRF2 protein levels, but did not induce telomere deprotection. Also in HeLa LT cells, induction of mitotic arrest induced Aurora B-dependent mitotic telomere deprotection, which corresponded with a reduction in looped telomere molecules. During mitotic arrest, inhibiting Aurora B rescued both DDR-activation, and the reduction of looped telomere molecules.  Cumulatively, our data indicate that t-loops function primarily to suppress DDR activation by the chromosome end.

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