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Biomedical subjects

D E Barnes

Publications and source records attributed to D E Barnes.

At least 19 recordsLinked to original sources
PubMed2001

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil-DNA glycosylase.

Gene-targeted mice deficient in the evolutionarily conserved uracil-DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(-) mutants. A complementary uracil-DNA glycosylase activity detected in ung(-/-) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil-DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.

Animals↗
PubMed2001

BRCT domain interactions in the heterodimeric DNA repair protein XRCC1-DNA ligase III.

Proteins involved in DNA repair, or its coordination with DNA replication and mitosis through cell cycle checkpoints, are vital in the concerted cellular response to DNA damage that maintains the integrity of the genome. The "BRCT" domain (BRCA1 carboxy terminal) was noted as a putative protein-protein interaction motif in the breast cancer suppressor gene, BRCA1, and subsequently identified in over 50 proteins involved in DNA repair, recombination, or cell cycle control. The heterodimer of the DNA repair proteins, XRCC1 and DNA ligase III, was the first example of a functional interaction via BRCT modules. The only three-dimensional crystal structure of a BRCT domain was solved for this region of XRCC1. Key amino acid residues mediating the interaction with DNA ligase III were identified here by targeted mutagenesis of the XRCC1 BRCT domain. The consequences of these mutations on protein folding were assessed. A structural model of the DNA ligase III BRCT domain was constructed and similarly tested by mutation of corresponding residues required for the interaction with XRCC1. These data identify the XRCC1-DNA ligase III heterodimer interface and provide the first demonstration of the surface contacts coordinating a functional BRCT-BRCT protein interaction.

Amino Acid Substitution↗
PubMed2001

Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice.

Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

Age Factors↗
PubMed2000

Defective neurogenesis resulting from DNA ligase IV deficiency requires Atm.

Ataxia telangiectasia results from mutations of ATM and is characterized by severe neurodegeneration and defective responses to DNA damage. Inactivation of certain DNA repair genes such as DNA ligase IV results in massive neuronal apoptosis and embryonic lethality in the mouse, indicating the occurrence of endogenously formed DNA double-strand breaks during nervous system development. Here we report that Atm is required for apoptosis in all areas of the DNA ligase IV-deficient developing nervous system. However, Atm deficiency failed to rescue deficits in immune differentiation in DNA ligase IV-null mice. These data indicate that ATM responds to endogenous DNA lesions and functions during development to eliminate neural cells that have incurred genomic damage. Therefore, ATM could be important for preventing accumulation of DNA-damaged cells in the nervous system that might eventually lead to the neurodegeneration observed in ataxia telangiectasia.

Animals↗
PubMed2000

Transcription coupled repair of 8-oxoguanine in murine cells: the ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences.

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7, 8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1(-/-) null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG.C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1(-/-) MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1(-/-) cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo.

Animals↗
PubMed2000

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication.

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

Animals↗
PubMed1999

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage.

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Animals↗
PubMed1998

Why review articles on the health effects of passive smoking reach different conclusions.

OBJECTIVE: To determine whether the conclusions of review articles on the health effects of passive smoking are associated with article quality, the affiliations of their authors, or other article characteristics. DATA SOURCES: Review articles published from 1980 to 1995 were identified through electronic searches of MEDLINE and EMBASE and from a database of symposium proceedings on passive smoking. ARTICLE SELECTION: An article was included if its stated or implied purpose was to review the scientific evidence that passive smoking is associated with 1 or more health outcomes. Articles were excluded if they did not focus specifically on the health effects of passive smoking or if they were not written in English. DATA EXTRACTION: Review article quality was evaluated by 2 independent assessors who were trained, followed a written protocol, had no disclosed conflicts of interest, and were blinded to all study hypotheses and identifying characteristics of articles. Article conclusions were categorized by the 2 assessors and by one of the authors. Author affiliation was classified as either tobacco industry affiliated or not, based on whether the authors were known to have received funding from or participated in activities sponsored by the tobacco industry. Other article characteristics were classified by one of the authors using predefined criteria. DATA SYNTHESIS: A total of 106 reviews were identified. Overall, 37% (39/106) of reviews concluded that passive smoking is not harmful to health; 74% (29/39) of these were written by authors with tobacco industry affiliations. In multiple logistic regression analyses controlling for article quality, peer review status, article topic, and year of publication, the only factor associated with concluding that passive smoking is not harmful was whether an author was affiliated with the tobacco industry (odds ratio, 88.4; 95% confidence interval, 16.4-476.5; P<.001). CONCLUSIONS: The conclusions of review articles are strongly associated with the affiliations of their authors. Authors of review articles should disclose potential financial conflicts of interest, and readers of review articles should consider authors' affiliations when deciding how to judge an article's conclusions.

Analysis of Variance↗
PubMed1997

XRCC1 protein interacts with one of two distinct forms of DNA ligase III.

Human DNA ligase III (103 kDa) has been shown to interact directly with the 70 kDa DNA repair protein, XRCC1. Here, the binding sites have been defined. Subcloned fragments of XRCC1 have been expressed and assayed for their ability to associate with DNA ligase III by far Western and affinity precipitation analyses. The C-terminal 96 amino acids of XRCC1 are necessary and sufficient for the specific interaction with DNA ligase III. A similar approach with the 103 kDa DNA ligase III has identified the C-terminal 148 amino acids of this enzyme as containing the binding site for XRCC1. An alternative 96 kDa form of DNA ligase III, abundant in testes, has been described [Chen, J., et al. (1995) Mol. Cell. Biol. 15, 5412-5422]. These two forms of DNA ligase III have identical N-terminal regions but differ toward their C termini and may be alternatively spliced products of the same gene. Antipeptide antibodies directed against the different C termini of the two forms of the enzyme indicate that both of them occur in vivo. The C-terminal region of the 96 kDa derivative of DNA ligase III is not able to interact with XRCC1. These findings indicate that only the larger form of DNA ligase III acts together with XRCC1, suggesting a role for this isoform of the enzyme in base excision repair.

Amino Acid Sequence↗
PubMed1997

Specific function of DNA ligase I in simian virus 40 DNA replication by human cell-free extracts is mediated by the amino-terminal non-catalytic domain.

The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I. This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro. Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable. Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts. The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the full-length protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication. Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain. This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.

Cell Line↗
PubMed1997

Repair and processing events at DNA ends.

Cell nuclei contain several abundant enzymes that bind rapidly and avidly to exposed termini of DNA. The properties and physiological roles of such factors are described; they include poly (ADP-ribose) polymerase, DNA-dependent protein kinase, several DNA ligases and excision-repair enzymes. Telomeres normally seem shielded from these activities by telomere-binding proteins. If incomplete protection of telomeres occurred, the functions of the DNA end-specific enzymes would be relevant for processing of telomeres. This could include alternative pathways for telomere propagation in telomerase-negative cells.

DNA↗
PubMed1997

Scientific quality of original research articles on environmental tobacco smoke.

OBJECTIVE: To evaluate the scientific quality of original research articles on the health effects of environmental tobacco smoke; to determine whether poor article quality is associated with publication in non-peer-reviewed symposium proceedings or with other article characteristics. DESIGN: Cross sectional study of original research articles on the health effects of environmental tobacco smoke published in peer reviewed journals and non-peer-reviewed symposium proceedings from 1980 to 1994. Article quality was assessed by two independent reviewers who used a valid and reliable instrument, were unaware of study hypotheses, were blinded to identifying characteristics of articles, and had no disclosed conflicts of interest. PARTICIPANTS: All symposium articles (n = 68) and a random sample of peer reviewed journal articles (n = 68) that satisfied inclusion/exclusion criteria. MAIN OUTCOME MEASURE: Mean quality scores, which could range from 0 (lowest quality) to 1 (highest quality). RESULTS: Using multivariate regression analysis, symposium articles were of poorer scientific quality than peer reviewed journal articles when controlling simultaneously for the effects of study design, article conclusion, article topic, and source of funding acknowledged (P = 0.027). Article quality was not associated with either source of funding acknowledged or article conclusion in multivariate analyses. CONCLUSIONS: In published reports on environmental tobacco smoke, non-peer-reviewed symposium articles tend to be of poor quality. These articles should not be used in scientific, legal, or policy settings unless their quality has been independently assessed.

Congresses as Topic↗
PubMed1996

Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.

Base Composition↗
PubMed1996

Industry-funded research and conflict of interest: an analysis of research sponsored by the tobacco industry through the Center for Indoor Air Research.

The Center for Indoor Air Research (CIAR) was created by three United States tobacco companies in 1988. Its stated mission is to fund high-quality, objective research related to indoor air, including studies of environmental tobacco smoke (ETS). Because CIAR is financed by the tobacco industry and funds research related to tobacco, it fosters an inherent conflict of interest. We consider whether this conflict of interest has affected the content, quality, or use of research funded by CIAR. We hypothesize that the tobacco industry might be using CIAR to develop scientific data to support its position that ETS is not harmful to health. CIAR funds two types of projects: "Peer-reviewed" projects are awarded after peer review by a group of scientists, whereas "special-reviewed" projects are awarded directly by tobacco industry executives. CIAR's special-reviewed projects are more likely than its peer-reviewed projects to be related to ETS, to support the tobacco industry position, and to be used by the industry to argue that smoking should not be regulated in public places. Our findings suggest that the tobacco industry is funding special-reviewed projects through CIAR to develop scientific data that it can use in legislative and legal settings. The industry may be financing peer-reviewed projects through CIAR to enhance its credibility, to provide good publicity, and to divert attention from ETS as an indoor air pollutant. CIAR's stated mission of funding high-quality, objective research has been compromised by conflict of interest, and at least some of CIAR's projects are being used to promote the tobacco industry's agenda.

Conflict of Interest↗
PubMed1995

Environmental tobacco smoke. The Brown and Williamson documents.

OBJECTIVE: To examine the tobacco industry's public and private responses to rising concern over the health effects of environmental tobacco smoke (ETS). DATA SOURCES: Documents from Brown and Williamson Tobacco Corporation (B&W), the British American Tobacco Company (BAT), and other tobacco interests provided by an anonymous source, obtained from Congress, and received from the private papers of a former BAT officer. STUDY SELECTION: All available materials, including confidential reports regarding research and internal memoranda exchanged between tobacco industry lawyers. CONCLUSIONS: Privately, B&W and BAT began conducting research related to ETS in the mid 1970s. BAT researchers appear to have determined that sidestream smoke produces irritation, that it contains toxic substances including N-nitrosamines, and that it is "biologically active" (eg, carcinogenic) in laboratory tests. During the 1980s, the primary purpose of BAT's research related to ETS was to develop a new cigarette that emitted less irritating and less biologically active sidestream smoke. Publicly, the tobacco industry has denied that exposure to ETS has been proven dangerous to health. It has criticized the methodology of published research on ETS, even when some of its own consultants have privately acknowledged that the research was valid. In addition, the industry has funded scientific research with the stated purpose of anticipating and refuting the evidence against ETS. The tobacco industry's strategy regarding passive smoking has been remarkably similar to its strategy regarding active smoking. It has privately conducted internal research, at least some of which has supported the conclusion that passive smoking is dangerous to health, while it has publicly denied that the hazards have been proven.

Humans↗