Publications by authors named "Jack D Griffith"

102 Publications

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions.

Elife 2021 Sep 8;10. Epub 2021 Sep 8.

Memorial Sloan Kettering Cancer Center, New York, United States.

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.
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http://dx.doi.org/10.7554/eLife.72286DOI Listing
September 2021

The mitochondrial single-stranded DNA binding protein is essential for initiation of mtDNA replication.

Sci Adv 2021 Jul 2;7(27). Epub 2021 Jul 2.

Department of Medical Biochemistry and Cell Biology, University of Gothenburg, PO Box 440, Gothenburg 405 30, Sweden.

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.
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http://dx.doi.org/10.1126/sciadv.abf8631DOI Listing
July 2021

Electron microscopic characterization of exhaust particles containing lead dibromide beads expelled from aircraft burning leaded gasoline.

Authors:
Jack D Griffith

Atmos Pollut Res 2020 Sep 4;11(9):1481-1486. Epub 2020 Jun 4.

Lineberger Comprehensive Cancer Center, Departments of Microbiology and Immunology, And Biochemistry and Biophysics University of North Carolina at Chapel Hill, Chapel Hill, NC, 27955, USA.

Piston powered aircraft burning leaded gasoline contribute ~70% of the lead in the atmosphere in the US. The physical size, composition, and structure of aircraft exhaust particles containing lead dibromide are poorly understood and heretofore have not been examined directly by electron microscopy (EM), in particular when captured from an aircraft in flight. To accomplish this, exhaust samples were trapped on EM supports within 10-15 ms of exiting the aircraft exhaust pipe. High angle annular detector dark field scanning EM revealed irregular particles with a mean diameter of 13 nm consisting of a 4 nm microcrystal of lead dibromide surrounded by a halo of hydrocarbons. In contrast, exhaust particles from an automobile burning leaded fuel averaged 35 nm in diameter and contained 5-10, 4 nm lead beads. Of significant concern, the smaller aircraft particles could penetrate mucosal barriers in the lung and be readily taken up by epithelial cells.
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http://dx.doi.org/10.1016/j.apr.2020.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583388PMC
September 2020

Twenty years of t-loops: A case study for the importance of collaboration in molecular biology.

DNA Repair (Amst) 2020 10 26;94:102901. Epub 2020 Jun 26.

Lineberger Comprehensive Cancer Center and Departments of Microbiology and Immunology, and Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. Electronic address:

Collaborative studies open doors to breakthroughs otherwise unattainable by any one laboratory alone. Here we describe the initial collaboration between the Griffith and de Lange laboratories that led to thinking about the telomere as a DNA template for homologous recombination, the proposal of telomere looping, and the first electron micrographs of t-loops. This was followed by collaborations that revealed t-loops across eukaryotic phyla. The Griffith and Tomáška/Nosek collaboration revealed circular telomeric DNA (t-circles) derived from the linear mitochondrial chromosomes of nonconventional yeast, which spurred discovery of t-circles in ALT-positive human cells. Collaborative work between the Griffith and McEachern labs demonstrated t-loops and t-circles in a series of yeast species. The de Lange and Zhuang laboratories then applied super-resolution light microscopy to demonstrate a genetic role for TRF2 in loop formation. Recent work from the Griffith laboratory linked telomere transcription with t-loop formation, providing a new model of the t-loop junction. A recent collaboration between the Cesare and Gaus laboratories utilized super-resolution light microscopy to provide details about t-loops as protective elements, followed by the Boulton and Cesare laboratories showing how cell cycle regulation of TRF2 and RTEL enables t-loop opening and reformation to promote telomere replication. Twenty years after the discovery of t-loops, we reflect on the collective history of their research as a case study in collaborative molecular biology.
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http://dx.doi.org/10.1016/j.dnarep.2020.102901DOI Listing
October 2020

Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations.

Proc Natl Acad Sci U S A 2020 06 10;117(25):14127-14138. Epub 2020 Jun 10.

Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.
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http://dx.doi.org/10.1073/pnas.1921311117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321962PMC
June 2020

Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts.

Sci Rep 2020 03 27;10(1):5575. Epub 2020 Mar 27.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 27709, USA.

Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.
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http://dx.doi.org/10.1038/s41598-020-61477-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101401PMC
March 2020

A New View of the T-Loop Junction: Implications for Self-Primed Telomere Extension, Expansion of Disease-Related Nucleotide Repeat Blocks, and Telomere Evolution.

Front Genet 2019 14;10:792. Epub 2019 Aug 14.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.

Telomere loops (t-loops) are formed at the ends of chromosomes in species ranging from humans to worms, plants, and with genetic manipulation, some yeast. Recent studies demonstrated that transcription of telomeric DNA leads to highly efficient t-loop formation. It was also shown that both DNA termini are inserted into the preceding DNA to generate a highly stable t-loop junction. Furthermore, some telomeric RNA remains present at the junction, potentially acting as a plug to further protect and stabilize the t-loop. Modeling the loop junction reveals two mechanisms by which the canonical chromosomal replication factors could extend the telomere in the absence of telomerase. One mechanism would utilize the annealed 3' terminus as a replication origin. evidence for the ability of the t-loop to prime telomere extension using the T7 replication factors is presented. A second mechanism would involve resolution of the Holliday junction present in the t-loop bubble by factors such as GEN1 to generate a rolling circle template at the extreme terminus of the telomere. This could lead to large expansions of the telomeric tract. Here, we propose that telomeres evolved as terminal elements containing long arrays of short nucleotide repeats due to the ability of such arrays to fold back into loops and self-prime their replicative extension. In this view, telomerase may have evolved later to provide a more precise mechanism of telomere maintenance. Both pathways have direct relevance to the alternative lengthening of telomeres (ALT) pathway. This view also provides a possible mechanism for the very large repeat expansions observed in nucleotide repeat diseases such as Fragile X syndrome, myotonic dystrophy, familial amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). The evolution of telomeres is discussed in the framework of these models.
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http://dx.doi.org/10.3389/fgene.2019.00792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702307PMC
August 2019

Distinct Binding Modes of Vinculin Isoforms Underlie Their Functional Differences.

Structure 2019 10 15;27(10):1527-1536.e3. Epub 2019 Aug 15.

Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Departments of Pharmacology and Departments of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA. Electronic address:

Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin in its ability to bundle filamentous actin (F-actin). To elucidate the molecular basis for these differences, we employed computational and experimental approaches. Results from these analyses suggest that the C terminus of both vinculin and metavinculin form stable interactions with the F-actin surface. However, the metavinculin tail (MVt) domain contains a 68 amino acid insert, with helix 1 (H1) sequestered into a globular subdomain, which protrudes from the F-actin surface and prevents actin bundling by sterically occluding actin filaments. Consistent with our model, deletion and selective point mutations within the MVt H1 disrupt this protruding structure, and facilitate actin bundling similar to vinculin tail (Vt) domain.
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http://dx.doi.org/10.1016/j.str.2019.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774862PMC
October 2019

Extracellular vesicles from Kaposi Sarcoma-associated herpesvirus lymphoma induce long-term endothelial cell reprogramming.

PLoS Pathog 2019 02 4;15(2):e1007536. Epub 2019 Feb 4.

Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Extracellular signaling is a mechanism that higher eukaryotes have evolved to facilitate organismal homeostasis. Recent years have seen an emerging interest in the role of secreted microvesicles, termed extracellular vesicles (EV) or exosomes in this signaling network. EV contents can be modified by the cell in response to stimuli, allowing them to relay information to neighboring cells, influencing their physiology. Here we show that the tumor virus Kaposi's Sarcoma-associated herpesvirus (KSHV) hijacks this signaling pathway to induce cell proliferation, migration, and transcriptome reprogramming in cells not infected with the virus. KSHV-EV activates the canonical MEK/ERK pathway, while not alerting innate immune regulators, allowing the virus to exert these changes without cellular pathogen recognition. Collectively, we propose that KSHV establishes a niche favorable for viral spread and cell transformation through cell-derived vesicles, all while avoiding detection.
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http://dx.doi.org/10.1371/journal.ppat.1007536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361468PMC
February 2019

Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles.

J Extracell Vesicles 2018 30;7(1):1541396. Epub 2018 Nov 30.

Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, The University of North Carolina, Chapel Hill, NC, USA.

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.
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http://dx.doi.org/10.1080/20013078.2018.1541396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282418PMC
November 2018

Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading.

Cell Rep 2018 06;23(12):3419-3428

Howard Hughes Medical Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

DNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.
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http://dx.doi.org/10.1016/j.celrep.2018.05.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086610PMC
June 2018

Long repeating (TTAGGG) single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation.

J Biol Chem 2018 06 19;293(24):9473-9485. Epub 2018 Apr 19.

From the Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599,

Conformations adopted by long stretches of single-stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ∼150 nt and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3') , as well as the interrogation of its structure by EM and single-molecule magnetic tweezers (smMT). This repeat is of particular interest because it contains a run of three contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ∼12-fold. The bead-like structures were 5 and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ∼28 nm at forces between 6 and 12 pN, well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed.
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http://dx.doi.org/10.1074/jbc.RA118.002158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005428PMC
June 2018

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis.

Proc Natl Acad Sci U S A 2018 05 16;115(18):4661-4665. Epub 2018 Apr 16.

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

Aberrant accumulation of misfolded Cu, Zn superoxide dismutase (SOD1) is a hallmark of SOD1-associated amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disease. While recent discovery of nonnative trimeric SOD1-associated neurotoxicity has suggested a potential pathway for motor neuron impairment, it is yet unknown whether large, insoluble aggregates are cytotoxic. Here we designed SOD1 mutations that specifically stabilize either the fibrillar form or the trimeric state of SOD1. The designed mutants display elevated populations of fibrils or trimers correspondingly, as demonstrated by gel filtration chromatography and electron microscopy. The trimer-stabilizing mutant, G147P, promoted cell death, even more potently in comparison with the aggressive ALS-associated mutants A4V and G93A. In contrast, the fibril-stabilizing mutants, N53I and D101I, positively impacted the survival of motor neuron-like cells. Hence, we conclude the SOD1 oligomer and not the mature form of aggregated fibril is critical for the neurotoxic effects in the model of ALS. The formation of large aggregates is in competition with trimer formation, suggesting that aggregation may be a protective mechanism against formation of toxic oligomeric intermediates.
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http://dx.doi.org/10.1073/pnas.1800187115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939103PMC
May 2018

Nef Secretion into Extracellular Vesicles or Exosomes Is Conserved across Human and Simian Immunodeficiency Viruses.

mBio 2018 02 6;9(1). Epub 2018 Feb 6.

Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells. Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. Nef is dispensable for viral replication but essential for AIDS progression Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression.
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http://dx.doi.org/10.1128/mBio.02344-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801467PMC
February 2018

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase.

Nucleic Acids Res 2018 04;46(6):3034-3046

Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, USA.

Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization.
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http://dx.doi.org/10.1093/nar/gky094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887560PMC
April 2018

Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA.

Mol Cell 2018 01 28;69(1):9-23.e6. Epub 2017 Dec 28.

Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, 405 30 Gothenburg, Sweden. Electronic address:

How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3α (Top3α) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3α-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3α is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3α as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.
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http://dx.doi.org/10.1016/j.molcel.2017.11.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935120PMC
January 2018

DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

DNA Repair (Amst) 2017 12 28;60:77-88. Epub 2017 Oct 28.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Electronic address:

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919216PMC
December 2017

The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks.

J Biol Chem 2017 09 25;292(38):15611-15621. Epub 2017 Jul 25.

From the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295 and.

During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication.
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http://dx.doi.org/10.1074/jbc.M117.799064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612096PMC
September 2017

Defective postsecretory maturation of MUC5B mucin in cystic fibrosis airways.

JCI Insight 2017 03 23;2(6):e89752. Epub 2017 Mar 23.

Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina at Chapel Hill, North Carolina, USA.

In cystic fibrosis (CF), airway mucus becomes thick and viscous, and its clearance from the airways is impaired. The gel-forming mucins undergo an ordered "unpacking/maturation" process after granular release that requires an optimum postsecretory environment, including hydration and pH. We hypothesized that this unpacking process is compromised in the CF lung due to abnormal transepithelial fluid transport that reduces airway surface hydration and alters ionic composition. Using human tracheobronchial epithelial cells derived from non-CF and CF donors and mucus samples from human subjects and domestic pigs, we investigated the process of postsecretory mucin unfolding/maturation, how these processes are defective in CF airways, and the probable mechanism underlying defective unfolding. First, we found that mucins released into a normal lung environment transform from a compact granular form to a linear form. Second, we demonstrated that this maturation process is defective in the CF airway environment. Finally, we demonstrated that independent of HCO and pH levels, airway surface dehydration was the major determinant of this abnormal unfolding process. This defective unfolding/maturation process after granular release suggests that the CF extracellular environment is ion/water depleted and likely contributes to abnormal mucus properties in CF airways prior to infection and inflammation.
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http://dx.doi.org/10.1172/jci.insight.89752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358479PMC
March 2017

Telomere Recognition and Assembly Mechanism of Mammalian Shelterin.

Cell Rep 2017 01;18(1):41-53

Laboratory for Cell Biology and Genetics, Rockefeller University, New York, NY 10065, USA. Electronic address:

Shelterin is a six-subunit protein complex that plays crucial roles in telomere length regulation, protection, and maintenance. Although several shelterin subunits have been studied in vitro, the biochemical properties of the fully assembled shelterin complex are not well defined. Here, we characterize shelterin using ensemble biochemical methods, electron microscopy, and single-molecule imaging to determine how shelterin recognizes and assembles onto telomeric repeats. We show that shelterin complexes can exist in solution and primarily locate telomeric DNA through a three-dimensional diffusive search. Shelterin can diffuse along non-telomeric DNA but is impeded by nucleosomes, arguing against extensive one-dimensional diffusion as a viable assembly mechanism. Our work supports a model in which individual shelterin complexes rapidly bind to telomeric repeats as independent functional units, which do not alter the DNA-binding mode of neighboring complexes but, rather, occupy telomeric DNA in a "beads on a string" configuration.
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http://dx.doi.org/10.1016/j.celrep.2016.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225662PMC
January 2017

Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops.

Nucleic Acids Res 2016 Nov 7;44(19):9369-9380. Epub 2016 Sep 7.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA

The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves.
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http://dx.doi.org/10.1093/nar/gkw779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100571PMC
November 2016

Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins.

PLoS One 2016 21;11(4):e0154225. Epub 2016 Apr 21.

Department of Genetics, Comenius University in Bratislava, Faculty of Natural Sciences, Ilkovicova 6, 842 15, Bratislava, Slovak Republic.

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154225PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839565PMC
April 2017

Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

Biosci Rep 2015 Dec 8;36(1):e00288. Epub 2015 Dec 8.

Departments of Genetics and Biochemistry, Comenius University in Bratislava, Faculty of Natural Sciences, Mlynska dolina, Ilkovicova 6, 842 15 Bratislava, Slovak Republic

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species.
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http://dx.doi.org/10.1042/BSR20150275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725248PMC
December 2015

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA.

J Biol Chem 2015 Nov 7;290(48):28697-707. Epub 2015 Oct 7.

From the Institute of Biosciences and Medical Technology, University of Tampere, 33520 Tampere, Finland, the Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48823, and

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.
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http://dx.doi.org/10.1074/jbc.M115.673707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661385PMC
November 2015

Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity.

Cell Rep 2015 Jun 4;11(11):1749-59. Epub 2015 Jun 4.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. Electronic address:

MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveals that homologous recombination and non-homologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology—either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.
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http://dx.doi.org/10.1016/j.celrep.2015.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4481157PMC
June 2015

A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans.

PLoS Genet 2015 Feb 18;11(2):e1004985. Epub 2015 Feb 18.

Department of Biology and Interdepartmental Graduate Program in Genetics, Genomics and Bioinformatics, University of California Riverside, Riverside, California, United States of America.

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.
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http://dx.doi.org/10.1371/journal.pgen.1004985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334201PMC
February 2015

DNA replication catalyzed by herpes simplex virus type 1 proteins reveals trombone loops at the fork.

J Biol Chem 2015 Jan 3;290(5):2539-45. Epub 2014 Dec 3.

From the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295

Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.
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http://dx.doi.org/10.1074/jbc.M114.623009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317027PMC
January 2015

TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro.

Biochemistry 2014 Sep 21;53(34):5485-95. Epub 2014 Aug 21.

Curriculum in Genetics and Molecular Biology, University of North Carolina , Chapel Hill, North Carolina 27599, United States.

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.
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http://dx.doi.org/10.1021/bi5006249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151696PMC
September 2014

Top3α is required during the convergent migration step of double Holliday junction dissolution.

PLoS One 2014 2;9(1):e83582. Epub 2014 Jan 2.

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America ; Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.

Although Blm and Top3α are known to form a minimal dissolvasome that can uniquely undo a double Holliday junction structure, the details of the mechanism remain unknown. It was originally suggested that Blm acts first to create a hemicatenane structure from branch migration of the junctions, followed by Top3α performing strand passage to decatenate the interlocking single strands. Recent evidence suggests that Top3α may also be important for assisting in the migration of the junctions. Using a mismatch-dHJ substrate (MM-DHJS) and eukaryotic Top1 (in place of Top3α), we show that the presence of a topoisomerase is required for Blm to substantially migrate a topologically constrained Holliday junction. When investigated by electron microscopy, these migrated structures did not resemble a hemicatenane. However, when Blm is together with Top3α, the dissolution reaction is processive with no pausing at a partially migrated structure. Potential mechanisms are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083582PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879244PMC
November 2014
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