Urinary thymine dimers and 8-oxo-2'-deoxyguanosine in psoriasis.
(25/767)
Psoralen in conjunction with UVA (PUVA) is perhaps the most effective treatment for psoriasis. It is, however, a risk factor for skin cancer in these patients and there is a need to develop non-invasive assays reflective of treatment-induced DNA damage. We report here the assessment of two important lesions, thymine dimer (T<>T) and 8-oxo-2'-deoxyguanosine (8-OHdG), in the urine of psoriasis patients. It was found that, once corrected for urine concentration, the psoriatic group had significantly higher (P<0. 0001) urinary levels of thymine dimers compared to the control group. No significant differences in urinary 8-OHdG levels were noted between the psoriatic, atopic dermatitis and control groups. Therefore biomonitoring of therapy from the very start with this simple and non-invasive assay could perhaps be an effective measure of the risk involved with the treatment allowing optimization for minimal-risk therapy. (+info)
The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.
(26/767)
Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. (+info)
Yeast autonomously replicating sequence binding factor is involved in nucleotide excision repair.
(27/767)
Nucleotide excision repair (NER) in yeast is effected by the concerted action of a large complex of proteins. Recently, we identified a stable subcomplex containing the yeast Rad7 and Rad16 proteins. Here, we report the identification of autonomously replicating sequence binding factor 1 (ABF1) as a component of the Rad7/Rad16 NER subcomplex. Yeast ABF1 protein is encoded by an essential gene required for DNA replication, transcriptional regulation, and gene silencing. We show that ABF1 plays a direct role in NER in vitro. Additionally, consistent with a role of ABF1 protein in NER in vivo, we show that certain temperature-sensitive abf1 mutant strains that are defective in DNA replication are specifically defective in the removal of photoproducts by NER and are sensitive to killing by ultraviolet (UV) radiation. These studies define a novel and unexpected role for ABF1 protein during NER in yeast. (+info)
Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair.
(28/767)
After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li-Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G(1) arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li-Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-jun loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair. (+info)
Artificial and solar UV radiation induces strand breaks and cyclobutane pyrimidine dimers in Bacillus subtilis spore DNA.
(29/767)
The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity. Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation. While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y. Xue and W. L. Nicholson, Appl. Environ. Microbiol. 62:2221-2227, 1996). We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis. DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers. DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter ("UV-A sunlight") accumulated only single-strand breaks and double-strand breaks. Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used. Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment. (+info)
Functional significance and induction by solar radiation of ultraviolet-absorbing sunscreens in field-grown soybean crops.
(30/767)
Colorless phenylpropanoid derivatives are known to protect plants from ultraviolet (UV) radiation, but their photoregulation and physiological roles under field conditions have not been investigated in detail. Here we describe a fast method to estimate the degree of UV penetration into photosynthetic tissue, which is based on chlorophyll fluorescence imaging. In Arabidopsis this technique clearly separated the UV-hypersensitive transparent testa (tt) tt5 and tt6 mutants from the wild type (WT) and tt3, tt4, and tt7 mutants. In field-grown soybean (Glycine max), we found significant differences in UV penetration among cultivars with different levels of leaf phenolics, and between plants grown under contrasting levels of solar UV-B. The reduction in UV penetration induced by ambient UV-B had direct implications for DNA integrity in the underlying leaf tissue; thus, the number of cyclobutane pyrimidine dimers caused by a short exposure to solar UV-B was much larger in leaves with high UV transmittance than in leaves pretreated with solar UV-B to increase the content phenylpropanoids. Most of the phenylpropanoid response to solar UV in field-grown soybeans was induced by the UV-B component (lambda +info)
Transcription-coupled DNA repair in yeast transcription factor IIE (TFIIE) mutants.
(31/767)
We examined the role of yeast transcription initiation factor IIE (TFIIE) in eukaryotic transcription-coupled repair (TCR), the preferential removal of DNA damage from the transcribed strands of genes over non-transcribed sequences. TFIIE can recruit the transcription initiation/repair factor TFIIH to the RNA polymerase II (RNA pol II) initiation complex to facilitate promoter clearance. Following exposure to UV radiation, the RNA pol II elongation complex is blocked at sites of UV-induced DNA damage, and may be recognized by nucleotide excision repair proteins, thus enabling TCR. The TFA1 gene encodes the large subunit of TFIIE. We determined how DNA repair is affected by TFA1 conditional mutations. In particular, we find proficient TCR in a heat-sensitive tfa1 mutant at the non-permissive temperature during which growth is inhibited and overall RNA pol II transcription is reported to be inhibited. We demonstrate that transcription of the RPB2 gene was reduced, but readily detectable, in the heat-sensitive tfa1 mutant at the non-permissive temperature and thereby prove that TCR does occur in an expressed gene in the absence of TFIIE in vivo. We demonstrate that TCR occurs even at low levels of transcription. (+info)
DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer.
(32/767)
Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2. (+info)