BIOCHEMISTRY OF DNA REPAIR AND TRANSCRIPTION

DNA 修复和转录的生物化学

• 批准号: 6288739
• 负责人: VILHELM A. BOHR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6288739
• 关键词: DNA directed RNA polymerase; DNA repair; RNA biosynthesis; biochemistry; cell free system; enzyme activity; genetic transcription; phosphorylation; protein kinase; ultraviolet radiation

Summary of work: Cockayne syndrome (CS) belongs to the category of premature aging disaeses where the individuals appear much older than their chronological age. Cells from CS patients are sensitive to UV light, exhibit a delay in recovery of DNA and RNA synthesis following irradiation, and are defective in preferential repair and strand- specific re-pair of active genes. Complementation studies demonstrate at least two genes involved in CS, designated CSA and CSB. CSB protein, by sequen-ce comparison, belongs to the SNF2 family of proteins, which have roles in transcriptional regulation, chromosome stability and DNA repair. The cellular and molecular phenotype of CS include a significantly increased sensitivity to a number of DNA-damaging agents including UV irradiation. Studies in CS cells were initially confined to DNA repair in the general, overall genome, where no defect was found . However, CS cells are defective in the preferential repair of active genes and in the preferential repair of the transcribed strand of such genes. This defect in transcription coupled repair (TCR) in CS is not only found after UV exposure but also after exposure to certain forms of oxidative stress. Transfection of the CSB gene into hamster cells with the CS-B phenotype completely restores TCR and UV resistance to normal levels, demonstrating that the defect in TCR in CS-B is due to mutation in that gene. The complex clinical phenotype of CS, however, suggests that DNA repair may not be the primary defect. We have reported a defect in basal transcription in CS both in vivo and in vitro. This transcription defect is seen in CS-B lymphoblastoid cells and fibroblasts without any exposure to stress such as UV light. A previous study found that expression of a metalloprotease was reduced by 50% in CS cells, and recently it was reported that the purified CSB protein stimulates transcription, presumably as an elongation factor. We have used an in vitro assay to measure the incision event of the DNA repair process. During the first step of BER, there is an incision in DNA 5 to the lesion. The incision can be quantitated in cell extracts by using oligonucleotide duplexes that contain a single 8-oxoG lesion at a defined site. In primary CS-B cell lines we observe a deficiency in incision. This deficiency can be complemented by transfection of the CS1AN (CS-B) cell line with a plasmid containing the intact CSB gene, suggesting a role for CSB in the recognition of 8-oxoG. This is the first report of a general, global repair defect in CS-B. This deficiency in incision most likely reflects a decrement in the activity of the 8-oxoG glycosylase, and in support of this we detect lower levels of expression of the human OGG1 gene in CS-B cells than in normal cells. The expression of OGG1 is markedly higher in CS1AN cells transfected with the wild type CSB gene. This is a novel and not previously reported property of the CSB gene and it leads to the suggestion that the CSB protein is involved in the regulation of the OGG1 and perhaps other DNA repair genes. The CSB protein apparently functions at the crossroads of DNA repair and transcription. It has been reported to interact with the structure specific incision endonuclease XPG, CSA protein, and RNA polymerase II. It has considerable homology to the SWI/SNF complex, which in yeast is associated with RNA polymerase II. The SWI/SNF complex is involved in the initiation phase of the transcription process. We have also observed that CS-B cells appear to have a more open chromatin structure than normal cells, and this would be compatible with a function that involves a role in chromatin structural assembly. It would appear that the CSB protein has more than one function and is most likely involved in a large number of protein-protein interactions in transcription and repair pathways. One or more of these is likely to be very important for the assembly of the DNA repair and transcription factory at the nuclear matrix. This is supported by previous studies showing that CS-B cells are defective in the early, nuclear matrix associated DNA repair A functional analysis of the CSB gene has been undertaken in our laboratory to better understand the nature of the molecular deficiencies observed in CS. Mutants, generated by site-directed mutagenesis have been tested for genetic complementation of CSB null cell lines by cell viability and RNA synthesis recovery upon exposure to UV light and other genotoxic agents. Point mutations in ATPase motifs I and II of CSB dramatically reduce CSB function in vivo suggesting that ATP hydrolysis by CSB protein is required for transcription-coupled repair of DNA damage. This mutant also shows dramatically increased apoptosis, suggesting a role for the CS protein in the apoptotic pathway. In contrast to the ATPase point muta-tions, deletions in the conserved acidic domain do not appear to interfere with the repair capacity of CSB protein. This suggests that this domain may be conserved in the SWI-SNF family for some other function. Further studies are in progress to address other important functions of CSB as they relate to the structural domains of the protein.

工作总结：柯凯因综合征（CS）属于