Evidence for a functional interaction between yeast Pol ε and PCNA in vivo

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA replication requires protein coordination, notably involving proliferating cell nuclear antigen (PCNA), a sliding clamp.
• PCNA interacts with DNA polymerases delta (Pol δ) and epsilon (Pol ε), crucial for DNA synthesis.
• The in vivo role of the Pol ε-PCNA interaction, especially in leading strand synthesis, is not fully understood.

Purpose of the Study:

• To investigate the in vivo functional significance of the interaction between yeast Pol ε and PCNA.
• To elucidate the specific interaction sites and their importance for DNA replication.

Main Methods:

• Utilized AlphaFold for in silico modeling of PCNA and yeast Pol ε complex formation.
• Generated yeast strains with mutations disrupting identified Pol ε-PCNA interaction interfaces.
• Performed biochemical assays to validate interaction disruption and assess functional consequences in vivo.

Main Results:

• AlphaFold models predicted two interaction sites between Pol ε and PCNA: the P-domain and a PIP-box.
• Biochemical assays confirmed the PIP-box as critical and the P-domain as a secondary interaction site.
• Mutants lacking PCNA interaction showed no phenotype in wild-type yeast but exhibited slower growth with extended leading strand synthesis.

Conclusions:

• PCNA enhances the processivity of Pol ε in vitro and in vivo.
• The Pol ε-PCNA interaction is important for efficient leading strand synthesis under conditions of limited replication origins.