At the same time, replication protein A positions the structurespecific endonucleases at the appropriate sites for dual incision: XPG bases at the side and the ERCCXPF complex nucleotides of the lesion.In humans, genetic and cell biological evidence indicates that CSA and CSB play a key role in TCR, but their functions remain to be elucidated.It has been reported that CSA interacts with CSB and the p subunit of TFIIH in vitro. It has been shown that CSB interacts with RNA polymerase II in a complex containing DNA and nascent RNA in vitro. The resulting quaternary complex has been shown to have an ability to recruit TFIIH, suggesting that CSB would recruit the NER proteins in vivo when RNA polymerase II encounters the lesion on the transcribed strand protein, designated XAB. Furthermore, microinjection of antiXAB antibodies specifically inhibited transcription as well as TCR but not GGR, suggesting that XAB is a novel factor participating in TCR and transcription itself.Positive transformants were classified into several groups based on crosshybridization.Out of positive clones, belonged to the group of XAB.Almost all of the recombinant XAB were recovered in the pellet.To visualize the recombinant XAB, the gel was stained with CBBR and destained in water.The gel stripe containing the recombinant XAB was cut out and used for immunization.After extensive washing with NETN buffer, bound proteins were eluted by boiling in SDS sample buffer.The immunocomplexes were purified in NETN buffer as described above, and bound proteins were eluted by boiling in SDS sample buffer.UDS, RRS, and transcription levels were quantified by counting the autoradiographically <a href="http://www.thechemlibrary.com/archives/318"></a>
induced silver grains above the nuclei. Mutational and structural analyses suggest that TPR domains play a role in intra and intermolecular protein interactions. Sequence analysis revealed that XAB has motifs of the class I TPR covering most of the protein, suggesting that XAB may function as an important factor for proteincomplex formations in NER.The interaction with CSA prompted us to examine whether XAB interacts with CSB as well, since both CS proteins are specifically involved in the TCR pathway.This immunoprecipitated fraction also contained a significant proportion of RNA polymerase II, as we have previously shown. Previously, we have shown that CSB together with RNA polymerase II is a part of a large protein complex. RNA polymerase II large subunit was detected in the immunoprecipitated fraction with antiXAB antiserum but absent in the fraction with control serum. . A novel protein, XAB, interacts with XPA.A, yeast twohybrid assay showing a specific interaction of XPA and XAB.The enzyme activities measured by a quantitative liquid assay are shown in the table on the right.These findings provide evidence that XAB interacts with the CSBRNA polymerase II complex in vivo.To further analyze the XAB function in living cells, we examined the effect of microinjected antiXAB antisera on various NER parameters.Dark shading, amino acid residues found in more than eight out of the repeats; light shading, conservative substitutions found in more than seven of the repeats.Gaps in the sequence alignments are indicated by dashes.The sizes and positions of molecular weight markers are shown at the top of the blots.