of work: Cockayne syndrome (CS) is a rare human disease that is characterized by arrested post-natal growth and resulted in premature aging and death. Cells from CS individuals are abnormally sensitive to killing by ultraviolet radiation as well as certain so-called UV-mimetic chemicals, such as 4-nitroquinoline-1-oxide and N-acetoxy-2-acetylaminofluorene. This cellular phenotype prompted extensive studies on the ability of CS cells to carry out nucleotide excision repair both in intact cells and in cell-free systems. Most conventional assays, including the use a cell-free system that supports transcription-independent nucleotide excision repair, indicate no defect in CS cells. However, CS cells are defective in the enhanced rate of repair of the template (transcribed) strand relative to the coding (non-transcribed) strand of transcriptionally active genes. In recent experiments from this laboratory we have demonstrated that mutations in the CSB gene are the cause of the transcription coupled repair defect. In hamster cells homologous to CSB we can transfect with a normal CSB gene and complement the repair defect. The mechanism of TCR in eukaryotes remains to be elucidated, and the CSB protein appears. These observations and discovery of dual function of transcription factor II H (TFIIH) in transcription and DNA repair, led to the """"""""transcriptional hypothesis"""""""" which postulates that transcription defects are the underlying basis for the pathology in some human diseases including Cockayne syndrome. In this laboratory we were interested to test this hypothesis experimentally, and we have demonstrated the reduced level of RNA polymerase II (Pol II) transcription in intact and permeabilized CS-B cells. The molecular mechanism responsible for this deficiency was further investigated in a cell-free system. We utilized in vitro transcription assay and determine Pol II transcription activity in extracts prepared from different CS cell lines in comparison to extracts prepared from normal cells. We found that in vitro transcription is CS extracts is highly sensitive to minor damages in template DNA arising during purification. This deficiency may be complemented by transfection of a CS-B cell line with normal CSB gene. Studies of transcription in vitro in a plasmid based system demonstrate a significant transcription defect in CSB cells. This defect may be related to oxidation damage or structural changes in the DNA which somehow affects the transcription in CSB cells but not in normal cells. Experiments in intact cells also demonstrate a defect in basal transcription which can be complemented by transfection with the normal CSB gene. Further, these experiments suggest that CSB cells may have a defect in the assembly of the higher order chromatin structural organization in conjunction with transcription and DNA repair. This is supported by the observation that CSB chromatin is much more sensitive to detergent than normal chromatin.
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