Differential temperature-dependent multimeric assemblies of replication and repair polymerases on DNA increase...

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Accurate DNA replication and repair are crucial for genome stability.
• Sulfolobus solfataricus (Sso) possesses both a B-family replication polymerase (Dpo1) and a Y-family repair polymerase (Dpo4).
• Understanding polymerase complex formation is key to DNA maintenance.

Purpose of the Study:

• To investigate the oligomeric states and DNA binding of Sso Dpo1 and Dpo4.
• To determine how concentration and temperature influence polymerase-DNA interactions.
• To elucidate the thermodynamic selection mechanisms for polymerase binding.

Main Methods:

• Protein cross-linking
• Isothermal titration calorimetry
• Analytical ultracentrifugation
• Temperature-dependent fluorescence anisotropy equilibrium binding experiments

Main Results:

• Confirmed a novel dimeric Dpo4 complex bound to DNA.
• Separated and quantified discrete binding events for trimeric Dpo1 and dimeric Dpo4 formation.
• Demonstrated temperature-dependent binding equilibria, favoring higher temperatures.
• Observed enhanced nucleotide incorporation processivity for oligomeric forms at higher temperatures.
• Showcased thermodynamic selection favoring Dpo1 trimer over Dpo4 dimer at high temperatures.

Conclusions:

• Oligomeric assembly of Dpo1 and Dpo4 on DNA is concentration- and temperature-dependent.
• Temperature influences polymerase binding and function, favoring specific oligomeric states.
• These findings provide insights into the discrimination mechanisms of DNA polymerases for genome maintenance.