Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• DNA replication fidelity is crucial for genomic stability.
• Replicative DNA polymerases possess 3"-5" exonuclease activity for proofreading, excising misincorporated nucleotides.
• The mechanism of primer strand transfer from the polymerization site to the exonuclease site remains poorly understood.

Purpose of the Study:

• To elucidate the molecular mechanism of primer strand transfer during proofreading by E. coli DNA polymerase Pol III.
• To identify the structural intermediates and kinetic pathways involved in the switch between polymerization and exonuclease states.

Main Methods:

• Utilized molecular simulations based on cryo-electron microscopy (cryo-EM) structures.
• Integrated biochemical analyses and targeted mutational experiments.
• Delineated the free energy path connecting polymerization and exonuclease states.

Main Results:

• Identified stable intermediate structures during primer strand transfer.
• Revealed conserved DNA polymerase Pol III residues stabilizing the transitioning primer strand.
• Characterized switching kinetics on a timescale of tens of milliseconds.
• Unveiled a complete polymerization-to-exonuclease switching mechanism.

Conclusions:

• The study provides a comprehensive molecular mechanism for the pol-to-exo switch in DNA polymerase proofreading.
• Conserved residues play a critical role in stabilizing the primer strand during transfer.
• The findings advance our understanding of DNA replication fidelity and error correction.