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Of Amino Acid Metabolism”]

Many proteins involved in transcriptional regulation bind to the CTD, and it appears likely that the phosphorylation status of the CTD helps determine which proteins will bind. The ubiquitinated residues are not located in the CTD.For individual experiments, the following agents were added to the cell culture media hbefore UV irradiation and were maintained in the media after irradiation: cycloheximide, mgml; and lactacystin, mM. Insoluble material was solubilized with SDSPAGE sample buffer, and equicellular amounts of TD extractable and unextractable material were subjected to immunoblot analysis.Panel A, UV radiation induces ubiquitination of the hyperphosphorylated. Repaircompetent fibroblasts were subjected to UV irradiation and allowed to recover at C for h.Similar results were obtained when cell extracts were normalized for total protein content. A, lanes, could be caused by the altered steadystate level of the protein or altered phosphorylation status of the CTD.The level of hypophosphorylated, which recognizes a nonCTD epitope. In addition, the same samples were immunoblotted with H, which is specific for the hyperphosphorylated as well as with H, indicating that the ubiquitinated forms were also hyperphosphorylated even though the forms whose steadystate level diminishes after UV irradiation were relatively hypophosphorylated. Repaircompetent and then UV irradiated and incubated at C in the presence of cycloheximide for the indicated times.An identical time course was also performed without cycloheximide. A UV time course performed in the presence of the protein synthesis inhibitor cycloheximide demonstrated that this is indeed the case. Panel B, XPD fibroblasts were treated with the proteasomal inhibitor MG at mM for hand then subjected to UV irradiation plus incubation at C in the presence of MG for the indicated time intervals. To control for the affect of lactacystin and MG, unirradiated samples were also collected at the htime points. Truncation of more than half of the CTD prevents its normal function, but truncation of the portion of the CTD including all lysine residues yields a molecule capable of supporting a viable cell. Pol II LS are nearly identical; there are only two sequence differences, neither of which involves CTD lysine residues. Each complementation group lacks a functional gene product required for a mechanistic step of NER. In all XP cells, ubiquitination and deubiquitination occurred to an extent similar to that seen in repaircompetent cells and with similar kinetics. Pol II LS is a component of the nuclear matrix, and its degradation may render DNA more susceptible to fragmentation of the sort observed during apoptosis. Furthermore, an antibody that recognizes a nonphosphorylated CTD epitope. Presumably, the number and identity of phosphorylated versus nonphosphorylated residues among the approximately serines, threonines, and tyrosines within the CTD determine the migration rate in SDSPAGE.The results presented here also establish that ubiquitination occurs on nonCTD lysine residues. Repaircompetent human fibroblasts or fibroblasts from individuals with the DNA repair deficiency syndrome XP were subjected to the indicated dose of UV radiation followed by hof recovery at C.Another recent study identified a specific ubiquitinprotein ligase. In repairdeficient cells or in those repaircompetent cells subjected to a lethal.

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