Screening for Repair-Deficient XPC Mutants

<div><p>(A) Time course of host cell reactivation assay. Human XP–C fibroblasts were transfected with an expression vector coding for wild-type XPC (pcXPC) or the empty control vector (pcDNA). Repair complementation was assessed after the indicated times by monitoring luciferase expression from a UV-irradiated reporter construct. Excision is reported as the percentage of wild-type activity after 15 h of incubation (± SD).</p> <p>(B) Specificity of the repair assay. XP–C fibroblasts were transfected with expression vectors coding for wild-type XPC (pcXPC or pXPC–GFP), wild-type XPA (pcXPA), the control vectors (pcDNA and pGFP), or vectors containing the Trp690Ser mutant sequences (pcW690S or pW690S–GFP). Excision is reported as the percentage of wild-type activity (15-h incubations).</p> <p>(C) Deletion of aromatic side chains. XP–C fibroblasts were transfected with vector pcXPC carrying the indicated mutations. DNA repair is expressed as the percentage of wild-type complementation (15-h incubations) after deduction of background luciferase activity obtained with the control vector. The dashed line indicates a threshold of 50% reduction in repair activity.</p> <p>(D) Replacement of aromatic residues by amino acids of different properties.</p> <p>(E) Deletion of the positively charged side chains of conserved Lys or Arg residues.</p> <p>(F) Ala substitutions of absolutely conserved positions in the center of the DNA-binding domain.</p>