bims-tricox 2023-08-27 papers

Issue of 2023–08–27
three papers selected by
Yash Verma, University of Zurich

Life Sci Alliance. 2023 Nov;pii: e202301965. [Epub ahead of print]6(11):

Allotopic expression of COX6 elucidates Atco-driven co-assembly of cytochrome oxidase and ATP synthase.

Leticia Veloso R Franco, Chen-Hsien Su, Lorisa Simas Teixeira, Jhulia Almeida Clarck Chagas, Mario Henrique Barros, Alexander Tzagoloff.

The Cox6 subunit of Saccharomyces cerevisiae cytochrome oxidase (COX) and the Atp9 subunit of the ATP synthase are encoded in nuclear and mitochondrial DNA, respectively. The two proteins interact to form Atco complexes that serve as the source of Atp9 for ATP synthase assembly. To determine if Atco is also a precursor of COX, we pulse-labeled Cox6 in isolated mitochondria of a cox6 nuclear mutant with COX6 in mitochondrial DNA. Only a small fraction of the newly translated Cox6 was found to be present in Atco, which can explain the low concentration of COX and poor complementation of the cox6 mutation by the allotopic gene. This and other pieces of evidence presented in this study indicate that Atco is an obligatory source of Cox6 for COX biogenesis. Together with our finding that atp9 mutants fail to assemble COX, we propose a regulatory model in which Atco unidirectionally couples the biogenesis of COX to that of the ATP synthase to maintain a proper ratio of these two complexes of oxidative phosphorylation.

DOI: https://doi.org/10.26508/lsa.202301965

Methods Mol Biol. 2023 ;2967 209-221

Bacteria and Yeast Colony PCR.

Humberto Pereira, Paulo César Silva, Björn Johansson.

The bacteria Escherichia coli and the yeast Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E. coli is almost always used for fundamental DNA manipulation, while yeast is the simplest host system for studying eukaryotic gene expression and performing large-scale DNA assembly. Yeast expression studies may also require altering the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence, and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and by the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols.

Keywords: Colony; Direct lysis; Escherichia coli; PCR; Saccharomyces cerevisiae; Yeast

DOI: https://doi.org/10.1007/978-1-0716-3358-8_17

DNA (Basel). 2022 Dec;2(4): 264-278

An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA-Protein Cross-Links from Mammalian Cells.

Wenyan Xu, Linlin Zhao.

DNA-Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.

Keywords: DNA damage; DNA lesions; DNA repair; abasic sites; mitochondrial DNA (mtDNA); mitochondrial transcription factor A (TFAM); protein DNA adducts

DOI: https://doi.org/10.3390/dna2040019