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Importantly, the increased sensitivity of this region, which indicates the formation of an open region downstream the initiation site, was found in the presence of both wild type and mutant TFIIH containing an inactive XPD helicase subunit.Earlier stages of open complex formation, in which a region upstream of the start site is still open, were analyzed using template DNA that allows stalling of the polymerase after the formation of a nucleotide transcript. Again, no difference could be discerned when wild type or mutant TFIIH were used. DNA lesion was studied in detail using a specific and sensitive method that detects the nucleotide platinated oligonucleotides released by the NER system.We assessed the activity of the mutant TFIIH in NER using a fully reconstituted system. For the latter purpose WCE were prepared from two human repairdeficient cell lines, XPB. However, reaction mixtures containing TFIIH with mutant KR XPD subunit showed no detectable release of damaged oligonucleotide. We estimate from other experiments that the sensitivity of the assay would detect dual incision activity as low as of the wild type level.Mutant KR XPDTFIIH complex was, however, able to complement weakly an extract derived from XPB cells. Correction of the incision defect by the mutant complex is lower than correction by wild type TFIIH, presumably because exchange of TFIIH subunits is needed for complementation of extract containing mutated XPB with mutant XPD TFIIH complex.Consistent with this, when wild type or mutant complex was added to extracts from XPD cells, only the wild type was able to correct the incision defect. To establish whether mutant TFIIH allows the formation of uncoupled or incisions, the single lesion DNA substrate was labeled at the end with respect to the lesion and was incubated with purified components.This assay detects all incisions, arising during the dual incision reaction or as uncoupled or incisions. In the presence of wild type TFIIH, incisions were readily formed. However, neither nor incisions were detected when the reactions were performed with TFIIH containing mutant XPD subunit. As a positive control, uncoupled incisions were efficiently placed in the presence of EA mutant XPG protein. Taken together, these data indicate that the specific placement and efficient formation of both incisions around a lesion depend on ATP hydrolysis and the DNA helicase activity of the XPD subunit of TFIIH.The in vivo observed increase in UDS roughly correlates with the increase in DNA synthesis measured in the in vitro assay. We failed to detect any dominant effect exerted by the mutant protein on either RNA synthesis or UDS after injection of wild type fibroblasts. By using a monoclonal antibody raised against human XPD, a specific band with equal intensity corresponding to the expected molecular mass of XPD is observed in lanes containing lysates from UV cells transfected with wild type or mutant human cDNA.Expression of the cDNA encoding wild type and KR mutant proteins was analyzed by immunoblotting. A specific band was only observed in lysates containing the wild type and KR proteins in approximately equal amounts when compared with a lysate derived from immortalized wild type cells.

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