Structures of dNTP intermediate states during DNA polymerase active site assembly

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• DNA polymerases are crucial for DNA replication and repair.
• High-fidelity DNA synthesis relies on precise enzyme-substrate interactions and conformational changes.
• Previous structural studies of DNA polymerase β (pol β) primarily captured the closed conformation after nucleotide binding.

Purpose of the Study:

• To elucidate the structural mechanisms underlying nucleotide selection by DNA polymerase β.
• To characterize intermediate states of pol β during DNA synthesis prior to active site closure.
• To understand how pol β distinguishes between correct and incorrect incoming nucleotides.

Main Methods:

• X-ray crystallography was used to determine the structures of pol β ternary complexes.
• A destabilized closed complex approach allowed trapping of open conformation intermediates.
• Structures were obtained with both correct and incorrect incoming nucleotides.

Main Results:

• Unique ternary complex structures of pol β in an open conformation were determined.
• Watson-Crick hydrogen bonding is assessed upon initial complex formation, even in the open state.
• Intermediate nucleotide-bound states with partial metal coordination were observed.
• The correct nucleotide maintains Watson-Crick hydrogen bonds throughout intermediate states, unlike the incorrect nucleotide.
• The triphosphate moiety of the incoming nucleotide undergoes conformational rearrangement before active site closure.

Conclusions:

• Early assessment of Watson-Crick hydrogen bonding in the open conformation contributes to DNA synthesis fidelity.
• Intermediate structural states reveal a mechanism for preventing misinsertion before the polymerase active site fully closes.
• These findings provide insights into the dynamic process of nucleotide selection and fidelity enhancement in DNA polymerases.