Each of these mutants contains an intact nuclear localization signal. Mouse cells containing defective BRCA have been shown to be deficient in TCR. Although the nontranscribed strand shows the expected result, the transcribed strand also shows little repair. In contrast, cells containing BRCAD were able to undergo TCR.Within hafter exposure, a much greater proportion of DHFR is present in the bound fraction. Cells containing BRCAD undergo TCR approximately times faster than cells lacking functional BRCA. Although BRCA does not affect doublestranded break repair, cells lacking BRCA are deficient in their ability to perform TCR.These findings suggest that the interaction of BRCA with the transcription machinery are physiologically important.This work also suggests functional differences between BRCA and BRCA.Whereas BRCA predominantly affects TCR, cells lacking BRCA have been shown to be deficient in doublestranded break repair. Jensen and Jeffrey T. Holt J. Biol. Chem. doi. jbc. Access the most updated version of this article at http:www.jbc.orgcontent When a correction for this article is posted Click here to choose from all of JBCs email alerts This article cites references, of which can be accessed free at http:www.jbc.orgcontent.full.htmlreflist Downloadedfromhttp: www.jbc.orgbyguestonSeptember, Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA.To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single photoproduct and determined the rate of damage excision under a variety of reaction conditions.We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA.Under experimental conditions conducive to binding of either XPA RPA or XPC to damaged DNA, the rate of damage removal was about fold faster for reactions in which XPA RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA.We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.In human nucleotide excision repair, polypeptides in six repair factors act in concert to excise DNA damage in the form of nucleotide long oligomers. The excision reaction has been characterized extensively: the XPG endo<a href="http://inhibit09.online/archives/221"></a>
nuclease makes the incision. However, the critical step of damage recognition remains poorly understood.Three proteins have been implicated in damage recognition: XPA. All three proteins have been reported to have moderate preference for damaged DNA compared with undamaged DNA as tested by electrophoretic mobility shift assay, filter binding assay, or damaged DNA affinity chromatography.Furthermore, using a pulldown assay it was found that the combination of RPA XPA conferred increased selectivity for damaged DNA. These findings led to a model whereby the initial damage sensing was performed by XPA RPA, which subsequently recruited the other repair factors to the site of damage. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with U.