Reference #: WAT-1015-333021
Submit Date: 03/05/2002 06:15:45-0500

Inducible nucleotide excision repair of UV induced CPDs from the transcribed and non-transcribed regions of the S.cerevisiae MFA2 gene

ABSTRACT:
In this study, the effect of a prior UV irradiation on the removal of cyclobutane pyrimidine dimers (CPDs) from the transcribed strand (TS) and non-transcribed strand (NTS) of the MFA2 gene in haploid Saccharomyces cerevisiae cells was investigated. We examined events at nucleotide resolution in the upstream regulatory region and the transcribed region of this locus. In NER competent cells, the pre-irradiation with a dose of 20 J/m2 enhances the removal of CPDs induced by a second UV dose of 100 J/m2 in the TS and the NTS of MFA2 gene except for the CPDs in the region +258 to +298 in the NTS, where the enhanced repair was absent. No inducible repair was observed in rad9, rad24, rad16 and rad26 cells, indicating two checkpoint genes RAD9 and RAD24, the global repair gene RAD16 and the transcription coupled repair gene RAD26 are essential for inducible NER

Keywords: Inducible nucleotide excision repair, Saccharomyces cerevisiae, MFA2 , Cyclobutane pyrimidine dimers

Reference #: MCK-1017-355915
Submit Date: 03/28/2002 16:38:47-0500

Does the efficient repair of UV-induced DNA damage require the induction of p53-responsive gene products?

ABSTRACT:
The p53 tumour suppressor is a transcription factor that is expressed at low levels in actively growing cells. In response to many cellular stresses including UV light, p53 protein levels increase dramatically resulting in increased expression of target genes. It has also been clearly demonstrated that p53 regulates nucleotide excision repair and stimulates the ability to recover of mRNA synthesis. Taken together, one could speculate that p53 regulates these recovery processes following UV-irradiation. However, we find that the induction of p53-regulated genes occurs too slowly to account for the role of p53 in stimulating nucleotide excision repair and the recovery of mRNA synthesis following UV-irradiation. Importantly, we find that p53 affects the basal level of expression of many proteins. I propose that p53 is functional in unstressed cells and this activity of p53 primes cells to efficiently repair DNA damage-induced by agents such as UV light.

Keywords: p53, ultraviolet, transcription, DNA repair

Reference #: 083339
Submit Date:

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Reference #: HAN-1016-841757
Submit Date: 03/22/2002 17:38:25-0500

Control of nucleotide excision repair: Comparisons between E. coli, rodent cells and human cells.

ABSTRACT:
There are two sub-pathways of nucleotide excision repair, global genomic repair (GGR) and transcription coupled repair (TCR). TCR is selective for the transcribed DNA strand in expressed genes and it deals with lesions which arrest translocation of RNA polymerase II. However, there is also recent evidence for transcription coupled base excision repair of several oxidative DNA lesions, thymine glycol and 8-oxo-Guanine, that do not pose blocks to transcription in vitro. TCR appears to be constitutive, but efficient GGR of some lesions requires SOS-inducible genes in E. coli, or p53-transactivated genes in human cells. In human cells, but not in most rodent cells, p53 transactivates the gene for p48 which is involved in a DNA damage binding activity. Expression of p48 correlates with efficient GGR of cyclobutane pyrimidine dimers (CPDs). This accounts for the rodent DNA "repairadox", that although cultured rodent and human cells typically display similar clonal survival characteristics after UV exposure, the cells from mice, rats, and hamsters are generally deficient in repair of CPDs. An understanding of this issue is important because rodents are so widely employed as surrogates for humans in genetic toxicology. Although most of our studies have employed UV and assays for photoproducts, we have found that p53 also controls GGR of low levels of the carcinogenic DNA adducts of the polycyclic aromatic hydrocarbons, benzo(a)pyrene and benzo(g)chrysene. The health relevance of these results is highlighted by the fact that the measured adduct levels are comparable to those found in certain human populations, such as smokers. We have also studied control of excision repair in terminally differentiated human neurons, in which we find that GGR is suppressed, but that repair of both strands of expressed genes is maintained. Models for the regulation of GGR and TCR will be discussed.

Keywords: nucleotide excision repair, inducible genomic repair, mammalian cells, p53

Reference #: MUL-1017-992846
Submit Date: 04/05/2002 01:32:24-0500

Assembly of the nucleotide excision repair factors in vivo

ABSTRACT:
Nucleotide excision repair (NER) is a versatile and highly conserved repair system capable of removing a wide range of DNA lesions that distort the stacking of the DNA double helix. These lesions include ultraviolet (UV)-light induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). The repair reaction is carried out by a multiprotein complex that recognises the DNA damage, makes dual incisions bracketing the DNA lesion and releases the damaged DNA in the form of oligomers. The question as to whether NER is carried out by a preassembled repairosome rather than by sequential assembly of repair proteins, has been a matter of debate. We employed local UV irradiation and fluorescence antibody-labeling to visualize the assemblage of the NER complex in normal and repair deficient (xeroderma pigmentosum) human cells. Of the two putative damage recognition protein complexes XPA-RPA and XPC-hHR23B, only the latter appears to be essential for recruitment of TFIIH to UV-irradiated areas. These proteins form a stable complex together with the XPG endonuclease in the absence of XPA protein; in contrast, recruitment of the ERCC1-XPF endonuclease depends on the presence of the XPA protein. We conclude that the recruitment of repair proteins to the DNA lesion is fully dependent on XPC-hHR23B whereas XPA-RPA participates only in a late step of NER. These experiments do not exclude that other proteins might bind to UV photolesions prior to XPC-HR23B. Indeed, it appears, that UV-damaged DNA binding protein (DDB), a heterodimer of the p48 and p127 proteins, plays an important auxiliary role in the recognition of UV-lesions in vivo by XPC-hHR23B and binds to the lesions prior to XPC-hHR23B. In cells from XP-E patients lacking DDB, NER complex formation after UV exposure is significantly retarded compared to normal human cells, but not absent. Taking together, our findings suggest that NER is mediated by sequential assemblage of repair protein complexes at the site of the DNA lesions rather than by the action of a preassembled repairosome. Most likely, DDB is the first component to recognize the UV photolesions and strongly stimulates the assemblage of the other NER components.

Keywords: Nucleotide excision repair, Xeroderma Pigmentosum, UV light

Reference #: RAI-1017-511588
Submit Date: 03/30/2002 10:49:57-0500

UV-enhanced reactivation of UV-damaged viruses and reporter genes in mammalian cells.

ABSTRACT:
Three decades ago several laboratories reported that pretreatment of mammalian cells with radiation or certain chemical carcinogens results in an enhanced survival for a number of UV-irradiated nuclear replicating viruses. In analogy with the phenomenon of Weigle reactivation for bacteriophage and the "SOS" response in bacteria, this suggested the possible existence of inducible DNA repair in mammalian cells. More recently we have used a non-replicating recombinant adenovirus, Ad5HCMVspllacZ, which expresses the -galactosidase reporter gene, to examine inducible repair of UV-damaged DNA in human cells. Prior UV-irradiation of normal human fibroblasts with low UV fluences resulted in an enhancement of host cell reactivation (HCR) for expression of the UV-damaged reporter gene. The UV-enhanced HCR in normal cells was transient reaching a maximum when cells were infected between 12 and 24 h after UV (and scored at 24 h after infection) and disappearing when cells were infected at 72 h after UV. The transient UV-enhanced HCR response was also detected in transcription coupled repair (TCR) proficient xeroderma pigmentosum (XP) group C cells, but was delayed or absent in TCR deficient XP and Cockayne syndrome cells. The UV-enhanced HCR response was also absent in Li-Fraumeni syndrome (LFS) cells, SV40-transformed normal human fibroblasts and normal human fibroblasts expressing the human papilloma virus (HPV) E6 or E7 gene. Using a quantitative PCR technique we show that prior UV-irradiation of normal human fibroblasts results in an enhanced rate of removal of photoproducts from the UV-damaged reporter gene indicating the enhanced HCR for reporter gene expression results from UV-induced DNA repair. Since SV40 transformation and HPV E7 expression abrogates the retinoblastoma (pRb) proteins, whereas HPV E6 abrogates p53 and LFS cells express mutant p53, these results indicate that the UV-induced DNA repair in normal human fibroblasts involves p53, one or more of the pRb family of proteins and TCR. (Supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society)

Keywords: DNA repair, host cell reactivation, adenovirus, reporter gene