دورية أكاديمية
Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
| العنوان: | Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels. |
|---|---|
| المؤلفون: | Gorospe CM; Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden., Repolês BM; Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden., Wanrooij PH; Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden. paulina.wanrooij@umu.se. |
| المصدر: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2615, pp. 293-314. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press, |
| مواضيع طبية MeSH: | DNA, Mitochondrial*/genetics , Ribonucleotides*/metabolism, Mitochondria/metabolism ; Nucleotides ; DNA Replication |
| مستخلص: | 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. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| References: | Grossman LI, Watson R, Vinograd J (1973) The presence of ribonucleotides in mature closed-circular mitochondrial DNA. Proc Natl Acad Sci U S A 70(12):3339–3343. (PMID: 10.1073/pnas.70.12.33394202844427232) Wong-Staal F, Mendelsohn J, Goulian M (1973) Ribonucleotides in closed circular mitochondrial DNA from HeLa cells. Biochem Biophys Res Commun 53(1):140–148. (PMID: 10.1016/0006-291X(73)91412-54582369) Miyaki M, Koide K, Ono T (1973) RNase and alkali sensitivity of closed circular mitochondrial DNA of rat ascites hepatoma cells. Biochem Biophys Res Commun 50(2):252–258. (PMID: 10.1016/0006-291X(73)90833-44347514) Reijns MAM, Rabe B, Rigby RE et al (2012) Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell 149(5):1008–1022. (PMID: 10.1016/j.cell.2012.04.011225790443383994) Berglund A-K, Navarrete C, Engqvist MKM et al (2017) Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA. PLoS Genet 13(2):e1006628. (PMID: 10.1371/journal.pgen.1006628282077485336301) Wanrooij PH, Engqvist MKM, Forslund JME et al (2017) Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired. Proc Natl Acad Sci U S A 114(47):12466–12471. (PMID: 10.1073/pnas.1713085114291092575703314) Wanrooij PH, Tran P, Thompson LJ et al (2020) Elimination of rNMPs from mitochondrial DNA has no effect on its stability. Proc Natl Acad Sci 117(25):14306–14313. (PMID: 10.1073/pnas.1916851117325137277322039) Moss CF, Dalla Rosa I, Hunt LE et al (2017) Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model. Nucleic Acids Res 45(22):12808–12815. (PMID: 10.1093/nar/gkx1009291065965728394) Li Y, Breaker RR (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2γ-hydroxyl group. J Am Chem Soc 121(23):5364–5372. (PMID: 10.1021/ja990592p) Jaishree TN, van der Marel GA, van Boom JH et al (1993) Structural influence of RNA incorporation in DNA: quantitative nuclear magnetic resonance refinement of d(CG)r(CG)d(CG) and d(CG)r(C)d(TAGCG). Biochemistry 32(18):4903–4911. (PMID: 10.1021/bi00069a0277683912) Chiu H-C, Koh KD, Evich M et al (2014) RNA intrusions change DNA elastic properties and structure. Nanoscale 6(17):10009–10017. (PMID: 10.1039/C4NR01794C24992674) DeRose EF, Perera L, Murray MS et al (2012) Solution structure of the Dickerson DNA dodecamer containing a single ribonucleotide. Biochemistry 51(12):2407–2416. (PMID: 10.1021/bi201710q22390730) Wanrooij PH, Chabes A (2019) Ribonucleotides in mitochondrial DNA. FEBS Lett 121:5364. Clausen AR, Lujan SA, Bukholder AB et al (2015) Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22(3):185–191. (PMID: 10.1038/nsmb.2957256222954351163) Koh KD, Balachander S, Hesselberg JR et al (2015) Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA. Nat Methods 12(3):251–257. – 3 p following 257. (PMID: 10.1038/nmeth.3259256221064686381) Daigaku Y, Keszthelyi A, Muller CA et al (2015) A global profile of replicative polymerase usage. Nat Struct Mol Biol 22(3):192–198. (PMID: 10.1038/nsmb.2962256647224789492) Reijns MAM, Kemp H, Ding J et al (2015) Lagging-strand replication shapes the mutational landscape of the genome. Nature 518(7540):502–506. (PMID: 10.1038/nature14183256241004374164) Kreisel K, Engqvist MKM, Clausen AR (2017) Simultaneous mapping and quantitation of ribonucleotides in human mitochondrial DNA. J Visualized Exp: JoVE (129):1–11. Sparks JL, Chon H, Cerritelli SM et al (2012) RNase H2-initiated ribonucleotide excision repair. Mol Cell 47(6):980–986. (PMID: 10.1016/j.molcel.2012.06.035228641163470915) Southern E (2006) Southern blotting. Nat Protoc 1(2):518–525. (PMID: 10.1038/nprot.2006.7317406277) |
| فهرسة مساهمة: | Keywords: Alkaline gels; Alkaline hydrolysis; Denaturing gels; Ribonucleotides; Southern blot; rNMPs |
| المشرفين على المادة: | 0 (DNA, Mitochondrial) 0 (Ribonucleotides) 0 (Nucleotides) |
| تواريخ الأحداث: | Date Created: 20230222 Date Completed: 20230224 Latest Revision: 20230305 |
| رمز التحديث: | 20240628 |
| DOI: | 10.1007/978-1-0716-2922-2_21 |
| PMID: | 36807800 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1940-6029 |
|---|---|
| DOI: | 10.1007/978-1-0716-2922-2_21 |