Hypothesis

Prelamin A accumulation, through direct inhibition of CK2α, reduces RFC1 phosphorylation and promotes its proteasomal degradation, thereby reproducing the replication‑fork destabilization seen in progerin‑expressing cells. This mechanism is amplified in mechanically stressed tissues (e.g., vasculature) where nuclear strain increases prelamin A‑CK2α interaction, explaining the cell‑type‑specific vulnerability to genomic instability during physiological aging.

Mechanistic Basis

• Prelamin A binds CK2α with high affinity, lowering its kinase activity and altering its nuclear distribution 3. CK2α normally phosphorylates RFC1 on serine residues that protect the replication‑factor‑C complex from serine‑protease‑mediated turnover 1. When CK2 activity is dampened, RFC1 loses these stabilizing phosphates, becomes susceptible to proteases such as HTRA2, and is degraded, leading to fork stalling and increased ssDNA gaps.
• In tissues subjected to cyclic mechanical stretch (vascular smooth muscle, endothelium), nuclear envelope deformation enhances the proximity of prelamin A at the inner nuclear membrane to CK2α, strengthening the inhibitory interaction. This strain‑dependent effect creates a feedback loop where nuclear stress raises prelamin A levels, further suppresses CK2, and exacerbates RFC1 loss.
• Spermidine disrupts the prelamin A–CK2α complex, restoring CK2 kinase activity 3. By rescuing RFC1 phosphorylation, spermidine should specifically protect replication forks in prelamin A‑accumulating cells, independent of external replication stress.

Testable Predictions

1. Cells expressing inducible prelamin A will show decreased RFC1‑Ser phosphorylation, increased RFC1 ubiquitination, and shorter replication‑fork tracts measured by DNA fiber assay; these effects will be rescued by CK2α overexpression or spermidine treatment.
2. Vascular smooth muscle cells exposed to cyclic stretch will exhibit higher prelamin A‑CK2α co‑immunoprecipitation, lower RFC1 levels, and elevated γH2AX foci compared to static controls; knockdown of prelamin A or CK2α activation will normalize these phenotypes.
3. In aged mouse aortas, pharmacological inhibition of CK2 will phenocopy prelamin A‑induced fork instability, whereas spermidine administration will reduce RFC1 loss and improve fork progression without altering total lamin A levels.

Experimental Approach

• Generate a doxycycline‑inducible prelamin A‑FLAG construct in human fibroblasts and vascular smooth muscle cells. Treat with doxycycline, +/- CK2α overexpression plasmid, and +/- spermidine (10‑50 µM). Measure RFC1‑Ser phosphorylation by phospho‑specific western blot, RFC1 half‑life via cycloheximide chase, and fork dynamics using IdU/CldU labeling.
• Apply a flex‑cell strain system (10% elongation, 1 Hz) to mimic vascular pulsatility. Quantify prelamin A‑CK2α interaction by proximity ligation assay, assess RFC1 levels, and quantify DNA damage (53BP1 foci). Include controls with LMNA‑ZMPSTE24 double knockout to isolate prelamin A effects.
• Use aged (24‑month) C57BL/6 mice treated with spermidine (3 mM in drinking water) for 3 months. Isolate aortic nuclei, perform chromatin fractionation, and evaluate RFC1 abundance, CK2 activity, and replication‑fork integrity via single‑molecule analysis of replicated DNA (SMARD).

If prelamin A‑mediated CK2 inhibition drives RFC1 degradation and fork instability, rescuing CK2 activity or blocking the prelamin A–CK2α interaction should restore replication fork protection specifically in prelamin A‑rich, mechanically stressed tissues, thereby distinguishing this pathway from progerin‑PCNA sequestration and providing a mechanistic link between nuclear lamina dynamics and physiological aging.