Identification of DNA polymerase theta inactivation mechanism

A method for identifying a DNA polymerase theta (Pol θ) inhibitor that eliminates nucleophilicity of nucleophilic residue Lys2383 of Pol θ and inhibits or reduces both lyase and polymerase activities of Pol θ, a Pol θ inhibitor that modifies nucleophilic residue Lys2575 of Pol θ, or a Pol θ inhibitor that modifies nucleophilic residue Lys2577 of Pol θ, the method comprising:

• (a) contacting Pol θ with one or more candidate Pol θ inhibitors in a Pol θ reaction buffer to form a pre-incubation mixture;
• (b) incubating the pre-incubation mixture for a period of time at room temperature;
• (c) diluting the pre-incubation mixture with a solution containing a DNA substrate and nucleotide triphosphates in the Pol θ reaction buffer;
• (d) measuring fluorescence of the diluted pre-incubation mixture for a period of time;
• and
• (e) identifying:
• i) a Pol θ inhibitor that eliminates nucleophilicity of nucleophilic residue Lys2383 of Pol θ and inhibits or reduces both lyase and polymerase activities of Pol θ,
• ii) a Pol θ inhibitor that modifies nucleophilic residue Lys2575 of Pol θ, or
• iii) a Pol θ inhibitor that modifies nucleophilic residue Lys2577 of Pol θ.