Sustained active site rigidity during synthesis by human DNA polymerase μ

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA polymerase micro (Pol micro) is essential for nonhomologous end joining (NHEJ), a critical DNA repair pathway.
• Pol micro uniquely handles double-strand DNA breaks (DSBs) with unpaired 3' ends during NHEJ.

Purpose of the Study:

• To structurally characterize the catalytic cycle of Pol micro during single-nucleotide incorporation.
• To elucidate the molecular mechanisms underlying Pol micro's function in NHEJ.

Main Methods:

• X-ray crystallography was used to determine the structures of Pol micro throughout its catalytic cycle.
• Site-directed mutagenesis was employed to investigate the role of specific Pol micro regions.

Main Results:

• Pol micro exhibits minimal conformational changes in protein or DNA during dNTP binding and catalysis, unlike other DNA polymerases.
• A significant conformational change occurs earlier in the catalytic cycle, involving the repositioning of the flexible loop 1 region upon DNA binding.
• Mutations in loop 1 alter Pol micro's catalytic activity and impair its function in NHEJ.

Conclusions:

• The unique structural dynamics of Pol micro, particularly the role of loop 1, are key to its specialized function in repairing DSBs with unpaired 3' ends via NHEJ.
• Specific residues within loop 1 are critical for Pol micro's template-dependent catalytic activity in NHEJ.