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ssDNA and ssRNA Promote Phase Condensation of SAMHD1.

Brandon E Smith1, Ankita Pohnerkar1, Benjamin Orris1

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SAMHD1 protein condenses into liquid-like droplets with nucleic acids, regulated by GTP. This liquid-liquid phase separation (LLPS) has implications for cancer and viral infection therapies.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Cellular Biology
  • Structural Biology

Background:

  • SAMHD1 (SAM domain and HD domain-containing protein 1) is a dNTPase with diverse roles including viral restriction and DNA repair.
  • While SAMHD1's functions involve liquid-liquid phase separation (LLPS), its own phase separation properties were previously uncharacterized.
  • The protein's tetrameric structure is allosterically activated by GTP binding, influencing its interaction with nucleic acids.

Purpose of the Study:

  • To investigate the potential for SAMHD1 to undergo liquid-liquid phase separation (LLPS).
  • To characterize the conditions and domains involved in SAMHD1 phase separation.
  • To explore the biological relevance and therapeutic potential of SAMHD1 LLPS.

Main Methods:

  • Computational prediction of phase separation propensity using MolPhase.
  • In vitro phase separation assays using PEG 2000, ssDNA, and ssRNA.
  • Biochemical analysis of domain-specific contributions (SAM and CT domains) and ligand effects (GTP, dGTP).
  • In vivo microinjection experiments in human cells to observe SAMHD1-ssDNA condensates.

Main Results:

  • SAMHD1 exhibits a moderate phase separation probability (0.65), with SAM and CT domains predicted to be key.
  • In vitro, SAMHD1 formed liquid-like droplets with ssDNA/ssRNA in the presence of PEG 2000, disrupted by domain deletions or A1 site ligands.
  • SAMHD1-ssDNA condensates were observed within the nuclei of human cells, indicating biological relevance.

Conclusions:

  • SAMHD1 can undergo liquid-liquid phase separation (LLPS) under specific conditions, particularly in the presence of nucleic acids.
  • The SAM and CT domains are crucial for SAMHD1 LLPS, and A1 site ligands regulate these condensates.
  • SAMHD1 LLPS offers potential as a novel therapeutic target for cancer and viral infections.