Hypothesis

We'll test whether ectopic Ndt80 expression gives somatic cells a germline‑grade editing budget by boosting HDR, enhancing shelterin‑guided BER, and forming a GUNC‑like sequestration compartment.

Mechanistic Basis

• Ndt80 drives a transcriptional program that removes senescence‑associated factors and, as shown in yeast, extends mitotic lifespan[4].
• In germ cells, high‑fidelity HDR is favored over error‑prone NHEJ, producing structural variants with 13‑17 bp homology signatures[1].
• The shelterin complex gates repair at telomeres, enhancing polymerase β‑dependent base excision repair while suppressing deleterious pathways[2].
• Gametogenesis physically excludes aggregates, ecDNA and nucleolar excess into the GUNC, a non‑inherited compartment[3].
• We propose that Ndt80 upregulates intrinsically disordered proteins that phase‑separate into a membraneless GUNC‑like hub, capturing damaged cargo for asymmetric disposal during cell division.

Testable Predictions

1. Ectopic Ndt80 in murine mesenchymal stem cells will increase RAD51 foci and decrease 53BP1/NHEJ markers after ionizing radiation.
2. Treated cells will show longer telomeres and higher polymerase β activity at chromosome ends, reflecting enhanced shelterin‑guided BER.
3. Fluorescent reporters for protein aggregates and extracellular circular DNA will accumulate in a transient perinuclear compartment that is excluded from daughter cells.
4. Epigenetic clocks (e.g., Horvath) will shift backward by ≥10 % after four weeks of inducible Ndt80 expression.
5. Co‑knockdown of RAD51 will abolish the HDR increase and prevent age‑reversal phenotypes, falsifying the HDR‑dependence claim.

Experimental Design

• Generate a doxycycline‑inducible Ndt80 transgenic mouse line crossed with a mesenchymal stem‑cell‑specific Cre (Prx1‑CreERT2).
• We'll administer doxycycline for 2 weeks, then assess:
  • γ‑H2AX and RAD51/53BP1 immunofluorescence after 2 Gy IR (flow cytometry).
  • Telomere length by qFISH and TRF assay.
  • Polymerase β activity via in‑vitro extraction assay.
  • Aggregate load using GFP‑tagged polyQ reporter and ecDNA detection by rolling‑circle amplification.
  • Epigenetic age using Illumina EPIC array on sorted stem cells.
• Parallel in‑vitro CRISPRi knockdown of RAD51 to test necessity.

If the predictions hold, somatic cells can be grafted with a germline‑grade editing budget; failure of any core prediction would refute the hypothesis.