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Repolês, Bruno MarçalORCID iD iconorcid.org/0000-0001-5704-3093
Publications (3 of 3) Show all publications
Gorospe, C. M., Repolês, B. M. & Wanrooij, P. H. (2023). Determination of the ribonucleotide content of mtDNA using alkaline gels. In: Thomas J. Nicholls; Jay P. Uhler; Maria Falkenberg (Ed.), Mitochondrial DNA: methods and protocols (pp. 293-314). New York: Humana Press, 2615
Open this publication in new window or tab >>Determination of the ribonucleotide content of mtDNA using alkaline gels
2023 (English)In: Mitochondrial DNA: methods and protocols / [ed] Thomas J. Nicholls; Jay P. Uhler; Maria Falkenberg, New York: Humana Press, 2023, Vol. 2615, p. 293-314Chapter in book (Refereed)
Abstract [en]

Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.

Place, publisher, year, edition, pages
New York: Humana Press, 2023
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 2615
Keywords
Alkaline gels, Alkaline hydrolysis, Denaturing gels, Ribonucleotides, rNMPs, Southern blot
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-205504 (URN)10.1007/978-1-0716-2922-2_21 (DOI)36807800 (PubMedID)2-s2.0-85148677775 (Scopus ID)9781071629215 (ISBN)9781071629222 (ISBN)
Available from: 2023-03-15 Created: 2023-03-15 Last updated: 2023-07-11Bibliographically approved
Carvalho, G., Repolês, B. M., Mendes, I. & Wanrooij, P. H. (2022). Mitochondrial DNA Instability in Mammalian Cells. Antioxidants and Redox Signaling, 36(13-15), 885-905
Open this publication in new window or tab >>Mitochondrial DNA Instability in Mammalian Cells
2022 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 36, no 13-15, p. 885-905Article, review/survey (Refereed) Published
Abstract [en]

Significance: The small, multicopy mitochondrial genome (mitochondrial DNA [mtDNA]) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this important genome and that jeopardize mtDNA stability, including various lesions in the DNA template, topological stress, and an insufficient nucleotide supply.

Recent Advances: An ever-growing array of DNA repair or maintenance factors are being reported to localize to the mitochondria. We review current knowledge regarding the mitochondrial factors that may contribute to the tolerance or repair of various types of changes in the mitochondrial genome, such as base damage, incorporated ribonucleotides, and strand breaks. We also discuss the newly discovered link between mtDNA instability and activation of the innate immune response.

Critical Issues: By which mechanisms do mitochondria respond to challenges that threaten mtDNA maintenance? What types of mtDNA damage are repaired, and when are the affected molecules degraded instead? And, finally, which forms of mtDNA instability trigger an immune response, and how?

Future Directions: Further work is required to understand the contribution of the DNA repair and damage-tolerance factors present in the mitochondrial compartment, as well as the balance between mtDNA repair and degradation. Finally, efforts to understand the events underlying mtDNA release into the cytosol are warranted. Pursuing these and many related avenues can improve our understanding of what goes wrong in mitochondrial disease.

Place, publisher, year, edition, pages
Mary Ann Liebert, 2022
Keywords
mitochondrial DNA, genome instability, DNA replication
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-187332 (URN)10.1089/ars.2021.0091 (DOI)000669978100001 ()34015960 (PubMedID)2-s2.0-85130003932 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Society for Medical Research (SSMF)Åke Wiberg Foundation
Available from: 2021-09-08 Created: 2021-09-08 Last updated: 2022-06-09Bibliographically approved
Repolês, B. M., Gorospe, C. M., Tran, P., Nilsson, A. K. & Wanrooij, P. H. (2021). The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method. Mitochondrion (Amsterdam. Print), 61, 179-187
Open this publication in new window or tab >>The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method
Show others...
2021 (English)In: Mitochondrion (Amsterdam. Print), ISSN 1567-7249, E-ISSN 1872-8278, Vol. 61, p. 179-187Article in journal (Refereed) Published
Abstract [en]

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
DNA integrity, Long-range PCR, Mitochondrial DNA, mtDNA, Nuclease activity
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-189552 (URN)10.1016/j.mito.2021.10.005 (DOI)000717836700001 ()2-s2.0-85118478892 (Scopus ID)
Funder
Åke Wiberg Foundation, M20-0132Swedish Cancer Society, 19 0022 JIA, 190098 Pj 01 HSwedish Society for Medical Research (SSMF), S17-0023Swedish Research Council, 2019-01874
Available from: 2021-11-16 Created: 2021-11-16 Last updated: 2023-09-05Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-5704-3093

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