Bruce Demple - Academia.edu (original) (raw)

Papers by Bruce Demple

Molecular and Cellular Biology, Aug 1, 2008

Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision r... more Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase ␥ and interferes with SP-BER by covalently trapping polymerase ␥ during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.

Journal of Bacteriology, Oct 1, 1997

Escherichia coli K-12 strains are normally tolerant to n-hexane and susceptible to cyclohexane. C... more Escherichia coli K-12 strains are normally tolerant to n-hexane and susceptible to cyclohexane. Constitutive expression of marA of the multiple antibiotic resistance (mar) locus or of the soxS or robA gene product produced tolerance to cyclohexane. Inactivation of the mar locus or the robA locus, but not the soxRS locus, increased organic solvent susceptibility in the wild type and Mar mutants (to both n-hexane and cyclohexane). The organic solvent hypersusceptibility is a newly described phenotype for a robA-inactivated strain. Multicopy expression of mar, soxS, or robA induced cyclohexane tolerance in strains with a deleted or inactivated chromosomal mar, soxRS, or robA locus; thus, each transcriptional activator acts independently of the others. However, in a strain with 39 kb of chromosomal DNA, including the mar locus, deleted, only the multicopy complete mar locus, consisting of its two operons, produced cyclohexane tolerance. Deletion of acrAB from either wild-type E. coli K-12 or a Mar mutant resulted in loss of tolerance to both n-hexane and cyclohexane. Organic solvent tolerance mediated by mar, soxS, or robA was not restored in strains with acrAB deleted. These findings strongly suggest that active efflux specified by the acrAB locus is linked to intrinsic organic solvent tolerance and to tolerance mediated by the marA, soxS, or robA gene product in E. coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Dec 2, 2008

Exposure of Escherichia coli to superoxide-generating drugs, such as menadione or paraquat, uniqu... more Exposure of Escherichia coli to superoxide-generating drugs, such as menadione or paraquat, uniquely induces-40 proteins, nine of which are under the positive control of the soxR locus (at min 92). We report here that certain mutations at a separate locus that we have named soxQ (at min 34) confer some of the phenotypes seen in soxR-constitutive strains, including resistance to menadione. A previously known mutation called cfxB, identified through antibiotic resistance, is likely an allele of soxQ. The soxQl and cfxB mutations cause transcriptional activation of the genes that encode Mn-containing superoxide dismutase, glucose 6-phosphate dehydrogenase, and the soi-17119::1ac and soi-28::1ac fusions. These genes are also activated by soxR, but the soxQl and cfxB mutations increase the synthesis of seven other proteins not influenced by soxR. Moreover, the soxQland cfxB-dependent phenotypes do not depend on the soxR gene, and gene induction by soxR in response to redox stress does not depend on the soxQ locus. As well as increasing cellular resistance to some oxidants, the soxQl and cfxB mutations confer elevated resistance to various antibiotics, probably via diminished expression of outer membrane protein OmpF. The marAl multiple-antibiotic resistance mutation (also at min 34) behaves like a weak allele of soxQ but probably resides in a nearby gene that, with soxQ, is part of a regulatory complex. We propose that soxQ helps control some oxidative stress proteins as part of another regulon that responds to an unknown environmental signal.

Free Radical Biology and Medicine, 1990

Springer eBooks, 2009

Plants appear to generate reactive oxygen species (ROS) as signaling molecules to control various... more Plants appear to generate reactive oxygen species (ROS) as signaling molecules to control various fundamental processes. With this background, this review aims to highlight the involvement of ROS, and their possible interactions with nitric oxide (NO) and glutathione (GSH) in the symbiosis between rhizobia and leguminous plants. This compatible interaction, which is very important for sustainable agriculture, leads to the formation of a novel organ capable of fixing atmospheric nitrogen. ROS are involved in the early steps of the symbiotic interaction: their presence is essential for the development of optimal symbiosis and points to a signaling role for ROS during the symbiotic process. ROS may also regulate nodule function by interacting with NO.

Human Molecular Genetics, Dec 1, 1992

Abasic (AP) sites in DNA are produced spontaneously and by many genotoxic agents. The repair of s... more Abasic (AP) sites in DNA are produced spontaneously and by many genotoxic agents. The repair of such damages is initiated by AP endonucleases, which are evidently ubiquitous. We employed the recently cloned cDNA, APE, that encodes the major human AP endonuclease, to isolate large genomic fragments that contain the intact APE gene. The sequence of 3 kb encompassing APE was determined (GenBank Accession No. M99703). The APE gene contains four small introns (ranging 130 to 566 bp) and five exons, the first of which is untranslated. The 0.5 kb of DNA sequence upstream of APE did revealed only a possible CCAAT box, but no other regulatory sites or a TATA box, consistent with the constitutive expression of AP endonuclease activity observed in other studies. The location of APE in the human genome was mapped to chromosome 14, bands q11.2-12, by fluorescence in situ hybridization of metaphase cells with DNA from the genomic clones and subclones. Although this locus has not been associated causally with genetic diseases of DNA repair, some translocations that affect 14q11.2-12 could compromise APE and lead to genetic instability.

Journal of Biological Chemistry, Mar 1, 1995

Molecular Microbiology, Jun 1, 1996

SoxS is a transcriptional activator of oxidative stress genes in Escherichia coli. SoxS in witro ... more SoxS is a transcriptional activator of oxidative stress genes in Escherichia coli. SoxS in witro binds the promoters of soxRS-regulated genes such as mice zwf, nfo and sodA, forms multiple protein-DNA complexes, and recruits RNA polymerase to the promoters. E. coli Rob protein, with an N-terminus 55% identical to SoxS, was initially identified by its binding to the oriC replication origin, but Rob in witro binds some of the same promoters as SoxS and in wiwo activates some SoxSregulated genes. In this work we show that the multiple complexes with SoxS arise from the presence at least two independent binding sites in each of the micF and zwf promoters. SoxS and Rob each form only a single complex with a 20 bp DNA oligonucleotide corresponding to the region immediately upstream of the-35 element of the micF promoter. Methylation interference identified several conserved purine residues required for binding to micF and five other SoxS-binding sites. Together with binding studies using mutated oligonucleotides and published DNase I footprinting data, this information was used to form a consensus for SoxS sequence specificity: AN2GCAYN7CWA (where N is any base, Y is a pyrimidine, and W is A or T). The sequence requirements for Rob binding differed somewhat from those of SoxS. Using the SoxS-binding consensus, several genes potentially regulated by soxRS were identified in an E. coli genomic database; some of these genes have functions that might contribute to cellular resistance to oxidative stress.

Journal of Biological Chemistry, Jul 1, 1994

Annual Review of Genetics, Dec 1, 1991

... oxidative agents or antibiotics (44). Stationary-Phase/Starvation Response: The katF Regulon ... more ... oxidative agents or antibiotics (44). Stationary-Phase/Starvation Response: The katF Regulon The katF locus was identified originally via mutations that diminish catalase activity in E. coli (64). E. coli contains two catalases, the ...

During NASA’s Apollo missions, inhalation of dust particles from lunar regolith was identified as... more During NASA’s Apollo missions, inhalation of dust particles from lunar regolith was identified as a potential occupational hazard for astronauts. These fine particles adhered tightly to spacesuits and were brought accidentally into the living areas of the spacecraft. Apollo astronauts reported that exposure to the dust caused intense respiratory and ocular irritation. This problem is a potential challenge for the Artemis Program, which aims to return humans to the Moon for extended stays in this decade. Since lunar dust is “weathered” by space radiation, solar wind, and the incessant bombardment of micrometeorites, we investigated whether treatment of lunar regolith simulants to mimic space weathering enhanced their toxicity. Two such simulants were employed in this research, Lunar Mare Simulant-1 (LMS-1), and Lunar Highlands Simulant-1 (LHS-1), which were applied to human lung epithelial cells (A549). In addition to pulverization, previously shown to increase dust toxicity sharply,...

ISEE Conference Abstracts, 2014

Background: Fish consumption is recommended because of the high content of beneficial nutrients s... more Background: Fish consumption is recommended because of the high content of beneficial nutrients such as selenium (Se) and omega-3 fatty acids which may play a role in redox homeostasis. However, se...

Lunar dust was reported during the Apollo missions as a potential threat to astronauts during fut... more Lunar dust was reported during the Apollo missions as a potential threat to astronauts during future exploration of the Moon by humans. The planned lunar explorations starting in 2024 bring this problem to the forefront. Occupational dust exposure (e.g., in miners,) causes silicosis and related pulmonary disease, and increases the risk of lung cancer. Lunar regolith is composed of unique material found exclusively on the lunar surface, and this material appears to have physical and chemical properties that could exacerbate toxicity to humans compared to terrestrial dusts. Our previous work [1] demonstrated that grinding to reduce particle size and expose fresh surfaces strongly enhanced the cytotoxicity and genotoxicity (DNA-damaging) effects of NASA-generated lunar regolith simulants. That work also indicated that the capacity of various simulants to cause DNA damage or cell death was not well correlated with their oxidant-generating activity in aqueous solution. The grinding proce...

DNA Repair, 2018

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberran... more Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end-joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3' exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this

F1000 - Post-publication peer review of the biomedical literature, 2017

CONSPECTUS: Our cellular genome is continuously exposed to a wide spectrum of exogenous and endog... more CONSPECTUS: Our cellular genome is continuously exposed to a wide spectrum of exogenous and endogenous DNA damaging agents. These agents can lead to formation of an extensive array of DNA lesions including singleand double-stranded breaks, inter-and intrastrand cross-links, abasic sites, and modification of DNA nucleobases. Persistence of these DNA lesions can be both mutagenic and cytotoxic, and can cause altered gene expression and cellular apoptosis leading to aging, cancer, and various neurological disorders. To combat the deleterious effects of DNA lesions, cells have a variety of DNA repair pathways responsible for restoring damaged DNA to its canonical form. Here we examine one of those repair pathways, the base excision repair (BER) pathway, a highly regulated network of enzymes responsible for repair of modified nucleobase and abasic site lesions. The enzymes required to reconstitute BER in vitro have been identified, and the repair event can be considered to occur in two parts: (1) excision of the modified nucleobase by a DNA glycosylase, and (2) filling the resulting "hole" with an undamaged nucleobase by a series of downstream enzymes. DNA glycosylases, which initiate a BER event, recognize and remove specific modified nucleobases and yield an abasic site as the product. The abasic site, a highly reactive BER intermediate, is further processed by AP endonuclease 1 (APE1), which cleaves the DNA backbone 5′ to the abasic site, generating a nick in the DNA backbone. After action of APE1, BER can follow one of two subpathways, the short-patch (SP) or long-patch (LP) version, which differ based on the number of nucleotides a polymerase incorporates at the nick site. DNA ligase is responsible for sealing the nick in the backbone and regenerating undamaged duplex. Not surprisingly, and consistent with the idea that BER maintains genetic stability, deficiency and/or inactivity of BER enzymes can be detrimental and result in cancer. Intriguingly, this DNA repair pathway has also been implicated in causing genetic instability by contributing to the trinucleotide repeat expansion associated with several neurological disorders. Within this Account, we outline the chemistry of the human BER pathway with a mechanistic focus on the DNA glycosylases that initiate the repair event. Furthermore, we describe kinetic studies of many BER enzymes as a means to understand the complex coordination that occurs during this highly regulated event. Finally, we examine the pitfalls associated with deficiency in BER activity, as well as instances when BER goes awry.