Reactions were analyzed as described by polyacrylamide. M urea gel electrophoresis, after inactivation of RNA polymerase II by heating for min at C and alkaline phosphatase treatment. After this preincubation, nucleotides were added and incubated for min at C.After incubation for hat C, DNA was isolated, linearized, and analyzed by agarose gel electrophoresis.After nuclear injection of cDNA constructs, cells were incubated in normal culture medium for hto allow expression of the injected DNA molecules.Subsequently, UDS was determined after UVC irradiation, incubation forhin thymidinecontaining culture medium, fixation, and in situ autoradiography.RNA synthesis was determined by counting autoradiographic grains above the nuclei of injected cells, after labeling with uridine during a pulselabeling periodof hinnormal culture medium.After selection with G, single clones were isolated and selected for expression of wild type and mutant XPD by immunoblot analysis.UV survival was assayed using thymidine incorporation. After h, cells were irradiated, pulselabeled in thymidinecontaining culture medium for h, fixed, and subjected to in situ autoradiography. The protein level of the wild type and KR mutant appeared comparable.This indicates that addition of the HA epitope tag does not negatively influence the function of the XPD protein in NER.Identical or similar residues are underlined; residues of the consensus nucleotidebinding motif are boxed.Indicated is the introduced point mutation at position. B, immunoblot analysis of CHO UV transfectants using antiHA CA monoclonal antibodies.Lysates were obtained from untransfected, and cells transfected with human cDNA encoding XPDHA wild type and XPDHA KR protein.Indicated are the positions of the molecular mass markers and the TFIIH subunits identified on the basis of <a href="http://inhibit09.online/archives/217"></a>
apparent molecular mass.The band corresponding to the p subunit could not be assigned conclusively because antibodies raised against the human protein did not crossreact.This is in agreement with the finding that XPD resides in two complexes, TFIIH and XPDCAK. We were not able to separate efficiently XPDCAK and TFIIH by subsequent chromatography as their purification properties are very similar.However, since the staining patterns of the wild type and KR mutant fractions were almost identical, the activities of the HA fractions could be directly compared.The reduction we observe is similar to that seen using recombinant TFIIH lacking the XPD subunit. The DNA helicase activities were compared using a radiolabeled mer oligonucleotide annealed to singlestranded M DNA.Radiolabeled ATP was incubated in the presence of ng of M singlestranded DNA with increasing amounts of XPD complex, and the released phosphate was separated by thin layer chromatography.B, DNA helicase activity is absent in KR XPD complex.Helicase activity was measured in the presence of increasing quantities XPD complex as detected by the release of a radiolabeled mer oligonucleotide from singlestranded M DNA.C, CTD kinase activity is not affected by the KR mutation in XPD.Activity was measured in the presence of wild type or KR XPD complex using a peptide containing four repeats of the conserved repeat YSPTSPS of the large subunit of RNA polymerase II and incubated with radiolabeled ATP.However, within the limits of detection, the mutant KR XPD complex added in the same quantity appears to be completely deficient in the ability to release the labeled oligonucleotide.