No description available.

No description available.

This study demonstrates the use of an adenovirus vector system to study the effect of a DNA repair gene on untransformed human fibroblasts. The bacteriophage T4 pyrimidine dimer DNA glycosylase (den V) gene has been inserted into the E3 region of human adenovirus type 5. The resulting recombinant virus Ad Den V was determined to be producing correctly initiated RNA from the RSV 3' LTR promoter used in the den V expression cartridge inserted into the virus. The effect of the den V gene product on human fibroblasts 'liras examined by assaying for the percent host cell reactivation (%HCR) of Vag production for UV irradiated Ad Den V in comparison to that for a control virus. It was shown that the %HCR was significantly greater for Ad Den V as compared to the control virus in xeroderma pigmentosum (XP) cells. UV survival of adenovirus in XP cells exhibited a two component nature. Introduction of the den V gene into XP group A cells increased the D0 value of the first component of the viral survival curve to a level similar to that of XPC cells, which showed no change in this component irrespective of the presence of the den V gene. It has been suggested that the den V gene is able to partially complement the deficiency in some XP cells because of its small size, allowing it to gain access to the DNA damage site where as the cellular repair enzyme complex can not. Since XPC cells are proficient in their alteration of DNA secondary structure prior to DNA excision repair, these results are consistant with the hypothesis that the first component of UV viral survival curves reflects the pathway involved in accessing the damaged sites. The manuscript of a paper has been included as an appendix. The work theorizes on the origin of mammalian immune system diversity and bacteriophage lambda, and their possible relationship to prokaryotic DNA repair genes. / Thesis / Master of Science (MS)

No description available.

Deux phénomènes liés au TFIIH ont été étudiés : l'influence des mutations spécifiques dans la sous-unité XPD de TFIIH sur la réponse transcriptionnelle de certains gènes après l'irradiation UV, et l'interaction entre le TFIIH et la transcription des gènes de classe III. Une analyse détaillée de la dynamique du transcriptome a été effectuée pour la réponse des cellules humaines mutantes XP-D/CS à l'UV. Il a été démontré que la dysrégulation sélective observée de l’expression des gènes était liée à l'incapacité pour la ré-initiation transcriptionnelle et à l'hétérochromatinisation suivante, où l'histonedésacétylase SIRT1 a été identifiée comme le principal facteur. Son inhibition a permis de recouvrer l'expression normale d'un nombre substantiel des gènes affectés. Une étude de la participation pangénomique du coeur de TFIIH dans latranscription a découvert son association avec les gènes actifs de classe III. Cette association a été démontrée être indépendante de Pol II. Le coeur de TFIIH a été montré participer directement à la transcription effectuée in vitro par Pol III. / In this work, two TFIIH-related phenomena were investigated : the influence of specific mutations in TFIIH XPD subunits on the transcriptional response of different genes on UV irradiation and the interaction between TFIIH and transcription of class III genes. For the first time the detailed investigation of transcriptome dynamics was carried out for the response of XP-D/CS mutant human cells to UV-irradiation. The transcription regulation nature of the observed selective gene expression dysregulation was clearly observed. Its relation to failure of transcription re-initiation and consequentheterochromatisation was demonstrated. SIRT1 histone deacetylase was identified as the main driver of the repressive chromatin establishment on the certain genes upon UV. Inhibition of SIRT1 was found to recover normal expression of substantial number of affected genes. SIRT1 mediated mechanism was shown to be XP-D/CS specific. A potential link between this longevity related protein and progeria features of XP-D/CS mutants was hypothesised. Genome-wide study of the involvement of the core TFIIH in transcription revealed its association with active class III genes, not described previously. This association was demonstrated to be Pol II-independent. The core TFIIH was shown to be directly involved in Pol III mediated transcription in vitro.

The sensitivity of Xeroderma pigmentosa (XP) patients to sunlight has spurred the discovery and genetic and biochemical analysis of the eight XP gene products (XPA-XPG plus XPV) responsible for this disorder. These studies also have served to elucidate the nucleotide excision repair (NER) process, especially the critical role played by the XPA protein. More recent studies have shown that NER also involves numerous other proteins normally employed in DNA metabolism and cell cycle regulation. Central among these is ataxia telangiectasia and Rad3-related (ATR), a protein kinase involved in intracellular signaling in response to DNA damage, especially DNA damage-induced replicative stresses. This review summarizes recent findings on the interplay between ATR as a DNA damage signaling kinase and as a novel ligand for intrinsic cell death proteins to delay damage-induced apoptosis, and on ATR’s regulation of XPA and the NER process for repair of UV-induced DNA adducts. ATR’s regulatory role in the cytosolic-to-nuclear translocation of XPA will be discussed. In addition, recent findings elucidating a non-NER role for XPA in DNA metabolism and genome stabilization at ds-ssDNA junctions, as exemplified in prematurely aging progeroid cells, also will be reviewed.