Publications by authors named "Cristina A Nadalutti"

6 Publications

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Perspectives on formaldehyde dysregulation: Mitochondrial DNA damage and repair in mammalian cells.

DNA Repair (Amst) 2021 09 11;105:103134. Epub 2021 May 11.

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

Maintaining genome stability involves coordination between different subcellular compartments providing cells with DNA repair systems that safeguard against environmental and endogenous stresses. Organisms produce the chemically reactive molecule formaldehyde as a component of one-carbon metabolism, and cells maintain systems to regulate endogenous levels of formaldehyde under physiological conditions, preventing genotoxicity, among other adverse effects. Dysregulation of formaldehyde is associated with several diseases, including cancer and neurodegenerative disorders. In the present review, we discuss the complex topic of endogenous formaldehyde metabolism and summarize advances in research on fo dysregulation, along with future research perspectives.
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http://dx.doi.org/10.1016/j.dnarep.2021.103134DOI Listing
September 2021

Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction.

Curr Protoc Toxicol 2020 12;86(1):e99

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, Raleigh, North Carolina.

Several cell lines of different origin are routinely used in research and drug development as important models to study human health and disease. Studying cells in culture represents an easy and convenient tool to approach complex biological questions, but the disadvantage is that they may not necessarily reflect what is effectively occurring in vivo. Human primary cells can help address this limitation, as they are isolated directly from human biological samples and can preserve the morphological and functional features of their tissue of origin. In addition, these can offer more relevant data and better solutions to investigators because they are not genetically manipulated. Human foreskin tissue discarded after surgery, for instance, represents a precious source for isolating such cells, including human foreskin fibroblasts (FSK), which are used in several areas of research and medicine. The overall health of cells is determined by the mitochondria. Alterations of cellular metabolism and cell death pathways depend, in part, on the number, size, distribution, and structure of mitochondria, and these can change under different cellular and pathological conditions. This highlights the need to develop accurate approaches to study mitochondria and evaluate their function. Here, we describe three easy, step-by-step protocols to study cellular viability and mitochondrial functionality in FSK. We describe how to use circumcision tissue obtained from the clinic to isolate FSK cells by mechanical and enzymatic disaggregation, how to use a cationic dye, crystal violet, which is retained by proliferating cells, to determine cell viability, and how to prepare samples to assess the metabolic status of cells by evaluating different mitochondrial parameters with transmission electron microscopy. We have successfully used the approaches outlined here to recapitulate physiological conditions in these cells in order to study the effects of increased intracellular levels of formaldehyde. © 2020 U.S. Government. Basic Protocol 1: Isolation and maintenance of human primary foreskin fibroblasts (FSK) Basic Protocol 2: Determination of cell viability by crystal violet staining Basic Protocol 3: Transmission electron microscopy to study cellular damage and mitochondrial dysfunction.
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http://dx.doi.org/10.1002/cptx.99DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757388PMC
December 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

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
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