Characterizing PNKP Lysine Mutants

The coding DNA sequence (CDS) of the human PNKP (gene accession# NM_007254.4) was amplified using the Q5 hot start high fidelity DNA polymerase (New England Biolabs), with HEK293 genomic DNA as a template, and the amplified PCR fragment was cloned in pCDNA3 (containing an N-terminal FLAG tag) using HindIII–BamHI sites as described earlier (20 (link)). K142 and K226 were mutated to Arginine (K142R & K226R) or glutamine (K142Q & K226Q), individually, using the Q5 site-directed mutagenesis kit (New England Biolabs), according to the manufacturer's protocol. PCR primers (Integrated DNA Technologies, Inc.) for site-directed mutagenesis were designed using NEBaseChanger. The primers to introduce these mutations are listed in Table 2. A double mutant (K142R/K226R) was generated using the single mutant K142R as a template and then the K226R mutation was introduced in it. Similarly, a K142Q/K226Q double mutant was generated using K142Q as a template.