Probing conformational changes of human DNA polymerase lambda using mass spectrometry-based protein footprinting

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Full-length human DNA polymerase lambda (fPollambda) possesses unique N-terminal domains.
• Previous crystallographic studies suggested limited domain rearrangement during catalysis.

Purpose of the Study:

• To investigate solution-phase conformational changes of fPollambda during catalysis.
• To elucidate the role of active site residues in DNA polymerase lambda function.

Main Methods:

• Mass spectrometry-based protein footprinting to map surface accessibility of Arg residues.
• Comparison of free enzyme, enzyme*gapped DNA, and enzyme*gapped DNA*dNTP complexes.
• Site-directed mutagenesis and pre-steady-state kinetic analyses.

Main Results:

• fPollambda exhibits no major conformational changes in solution during catalysis.
• Active site residue R386 becomes shielded only when both gapped DNA and dNTP are bound.
• R386 is essential for enzyme activity, stabilizing nucleotide and pyrophosphate interactions.

Conclusions:

• DNA polymerase lambda functions without significant domain rearrangement in solution.
• Residue R386 plays a critical role in stabilizing charged intermediates during nucleotide incorporation.
• The catalytic mechanism involving R386 may be conserved across various DNA polymerases.