Break-induced replication is enhanced by a phospho-activated RPA-binding module in Pol32

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Homologous Recombination

Background:

• Break-induced replication (BIR) is crucial for repairing DNA double-strand breaks (DSBs) using homologous recombination.
• The minimal replisome for BIR includes PCNA, DNA polymerase δ (Pol δ), and Pif1 helicase.

Purpose of the Study:

• To identify and characterize the interaction between Pol δ and RPA during BIR.
• To elucidate the role of Pol32 phosphorylation in regulating BIR efficiency.

Main Methods:

• Protein interaction studies identifying an RPA-binding module (RBM) in Pol32.
• Site-directed mutagenesis and phospho-mimetic substitutions in Pol32.
• In vivo assessment of BIR efficiency.
• Molecular modeling of protein interactions.

Main Results:

• An RBM in Pol32 interacts with RPA subunit Rfa1.
• Phosphorylation of Pol32 RBM at Thr256/Thr257 enhances Rfa1 binding.
• Phospho-mimetic substitutions in Pol32 increase BIR efficiency in vivo.
• Modeling suggests conserved RBM-RPA interactions across related helicases.

Conclusions:

• Pol32 acts as a phosphorylation-dependent rheostat, modulating Pol δ affinity for RPA-bound BIR intermediates.
• RPA plays a central role in orchestrating the enzymatic machinery of BIR.
• Understanding these interactions provides insights into DNA repair pathway regulation.