p53-Dependent Regulation of Nucleotide Excision Repair in Murine Epidermis in vivo

1998 ◽  
Vol 3 (1) ◽  
pp. 16-20 ◽  
Author(s):  
Victor A. Tron ◽  
Martin J. Trotter ◽  
Takatoshi Ishikawa ◽  
Vincent C. Ho ◽  
Gang Li
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Mouse Skin ◽  
Computer Assisted ◽  
Pyrimidine Dimers ◽  
Cyclobutane Dimers ◽  
Uv Dose ◽  
Programed Cell Death

Background: p53 protects the integrity of the genome by inducing programed cell death or by promoting DNA repair. We have previously shown that loss or mutation of p53 leads to reduced DNA repair in keratinocytes. Objective: The hypothesis that p53 regulates repair of ultraviolet light-induced epidermal DNA damage in vivo was tested in mice. Methods: An immunohistochemical assay for pyrimidine dimers and 6–4 photoproducts was performed on ultraviolet-irradiated skin from p53 null (−/−) and wild type (+/+) mice. Immunostaining for photoproducts was quantified using computer-assisted imaging. The level of DNA repair was then expressed as the percentage of positive cells remaining as compared to the zero hour time point. Results: p53+/+ mouse skin exposed to 1000 J/m2 retained ≈ 25% of epidermal cyclobutane dimers at 48 h, whereas approximately 50% remained in p53−/− cells. Using the same UV dose, p53+/+ mice retained 20% of detectable 6–4 photoproducts by 24 h, whereas about 50% remained in epidermal cells of p53-deficient mice. Conclusion: Using in situ labelling of UV-damaged cells, we confirm our earlier conclusion that p53 regulates DNA repair within the epidermis after exposure to UV light.

scholarly journals The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

1999 ◽  
Vol 10 (11) ◽  
pp. 3583-3594 ◽  
Author(s):  
Robert M. Brosh ◽  
Adayabalam S. Balajee ◽  
Rebecca R. Selzer ◽  
Morten Sunesen ◽  
Luca Proietti De Santis ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Genetic Disorder ◽  
Cockayne Syndrome ◽  
Premature Aging ◽  
Atpase Domain ◽  
Acidic Region

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA andCSB, 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, theCSB 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.

scholarly journals An alternative eukaryotic DNA excision repair pathway.

1995 ◽  
Vol 15 (8) ◽  
pp. 4572-4577 ◽  
Author(s):  
G A Freyer ◽  
S Davey ◽  
J V Ferrer ◽  
A M Martin ◽  
D Beach ◽  
...  
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Repair Process ◽  
Dna Lesions ◽  
Repair Pathway ◽  
Cyclobutane Pyrimidine Dimers ◽  
Dna Excision Repair ◽  
Pyrimidine Dimers

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.

Preferential repair of damage in actively transcribed DNA sequences in vivo

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Philip C. Hanawalt
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Human Cells ◽  
Dhfr Gene ◽  
Pyrimidine Dimers ◽  
Cross Links

My colleagues and I have discovered intragenomic heterogeneity in DNA repair in mammalian cells. Consequences of unrepaired DNA damage depend upon the precise location of the damage with respect to relevant genes. It is therefore important to understand rules governing accessibility of specific DNA sequences in chromatin to damage and repair. The efficiency of removal of pyrimidine dimers has been determined in the active dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Repair within the gene was shown to be much more efficient than that in nontranscribed downstream sequences or in the genome overall. Preferential repair of active and essential genes such as DHFR may account for the fact that rodent cells are as uv-resistant as human cells in spite of their much lower overall repair efficiencies. In repair-proficient human cells the rate of repair in the DHFR gene is greater than that in the overall genome or in nontranscribed α-DNA sequences. The efficiency of removal of pyrimidine dimers is much higher in the transcribed than the nontranscribed DNA strands of the DHFR gene in both CHO and human cells. An excision–repair complex may be directly coupled to the transcription machinery to ensure early removal of transcription-blocking lesions in active genes. Sequences in the active c-abl proto-oncogene are repaired much more efficiently than are sequences containing the inactive c-mos proto-oncogene in Swiss mouse 3T3 cells. Tissue-specific and cell-specific differences in the coordinate regulation of proto-oncogene expression and DNA repair may account for corresponding differences in the carcinogenic response. Efficient replicative bypass of persisting psoralen monoadducts, but not interstrand cross-links, was demonstrated in the human DHFR gene. It is likely that most bulky lesions in mammalian DNA, other than cross-links, do not pose insurmountable problems for replication in vivo, but they must be removed from essential transcribed sequences to maintain cellular viability.Key words: DNA repair, chromatin, transcription, mammalian cells, pyrimidine dimers, ultraviolet light, DNA cross-links.

scholarly journals The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

1997 ◽  
Vol 17 (5) ◽  
pp. 2381-2390 ◽  
Author(s):  
A E Parker ◽  
R K Clyne ◽  
A M Carr ◽  
T J Kelly
Keyword(s):  
Dna Repair ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

scholarly journals The Dark Side of UV-Induced DNA Lesion Repair

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1450
Author(s):  
Wojciech Strzałka ◽  
Piotr Zgłobicki ◽  
Ewa Kowalska ◽  
Aneta Bażant ◽  
Dariusz Dziga ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Uv Light ◽  
Genetic Material ◽  
Dna Lesions ◽  
Dark Side ◽  
Repair System ◽  
Strand Breaks ◽  
Dna Lesion ◽  
Pyrimidine Dimers

In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers. In this review, we have focused on how plants cope with deleterious DNA damage that cannot be repaired by photoreactivation. The current understanding of light-independent mechanisms, classified as dark DNA repair, indispensable for the maintenance of plant genetic material integrity has been presented.

scholarly journals Databases and Bioinformatics Tools for the Study of DNA Repair

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Kaja Milanowska ◽  
Kristian Rother ◽  
Janusz M. Bujnicki
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
Environmental Chemicals ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Repair Mechanisms ◽  
Base Excision

DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.

scholarly journals Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

2000 ◽  
Vol 23 (3) ◽  
pp. 689-694
Author(s):  
E.N. Miyaji ◽  
R.T. Johnson ◽  
C.S. Downes ◽  
E. Eveno ◽  
M. Mezzina ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Excision Repair ◽  
Uv Light ◽  
Cell Metabolism ◽  
Cell Cycle Control ◽  
Pyrimidine Dimers ◽  
Dna Replication And Repair ◽  
Increased Sensitivity

Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2) that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, possibly indicating a defect in preferential repair of actively transcribed genes, and a slower cell proliferation rate, including a longer S-phase. This phenotype reinforces the present notion that control of key mechanisms in cell metabolism, such as cell cycle control, repair, transcription and cell death, can be linked.

scholarly journals Growth Retardation, Early Death, and DNA Repair Defects in Mice Deficient for the Nucleotide Excision Repair Enzyme XPF

2004 ◽  
Vol 24 (3) ◽  
pp. 1200-1205 ◽  
Author(s):  
Ming Tian ◽  
Reiko Shinkura ◽  
Nobuhiko Shinkura ◽  
Frederick W. Alt
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Uv Irradiation ◽  
Excision Repair ◽  
Genetic Defect ◽  
Growth Defect ◽  
Human Genetic Disease ◽  
Nucleotide Excision ◽  
Deficient Mice

ABSTRACT Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5′ to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.

scholarly journals True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Nicolas Le May ◽  
Jean-Marc Egly ◽  
Frédéric Coin
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Genetic Disorders ◽  
Accelerated Aging ◽  
Synthesis Process ◽  
Developmental Defects ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision ◽  
Active Genes

Nucleotide excision repair (NER) is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45αprotein (Gadd45α) and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

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