bims-mitran 2022-10-16 papers

Issue of 2022‒10‒16
three papers selected by
Andreas Kohler

Nat Biotechnol. 2022 Oct 13.

Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors.

Seonghyun Lee, Hyunji Lee, Gayoung Baek, Jin-Soo Kim.

Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.

DOI: https://doi.org/10.1038/s41587-022-01486-w

Nucleic Acids Res. 2022 Oct 10. pii: gkac857. [Epub ahead of print]

Two type I topoisomerases maintain DNA topology in human mitochondria.

Katja E Menger, James Chapman, Héctor Díaz-Maldonado, Mushtaq M Khazeem, Dasha Deen, Direnis Erdinc, John W Casement, Valeria Di Leo, Angela Pyle, Alejandro Rodríguez-Luis, Ian G Cowell, Maria Falkenberg, Caroline A Austin, Thomas J Nicholls.

Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.

DOI: https://doi.org/10.1093/nar/gkac857

J Biol Chem. 2022 Oct 06. pii: S0021-9258(22)01018-3. [Epub ahead of print] 102574

Precise and Simultaneous Quantification of Mitochondrial DNA Heteroplasmy and Copy Number by Digital PCR.

Wendy K Shoop, Cassandra L Gorsuch, Sandra R Bacman, Carlos T Moraes.

Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases. However, current methods can be imprecise, and quantifying small changes in either heteroplasmy or copy number is challenging. We developed a new approach to measure mtDNA heteroplasmy using a single digital PCR (dPCR) probe. This method is based on the observation that fluorescent-labeled probes in dPCR exhibit different intensities depending on the presence of a single nucleotide change in the sequence bound by the probe. This finding allowed us to precisely and simultaneously determine mtDNA copy number and heteroplasmy levels using duplex dPCR. We tested this approach in two different models (human and mouse), which proved faster and more internally controlled when compared to other published methods routinely used in the mitochondrial genetics field. We believe this approach could be broadly applicable to the detection and quantification of other mixed genetic variations.

DOI: https://doi.org/10.1016/j.jbc.2022.102574