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Viruses have developed sophisticated biochemical and genetic mechanisms to manipulate and exploit their hosts. Enzymes derived from viruses have been essential research tools since the first days of molecular biology. However, most viral enzymes that have been commercialized are derived from a small number of cultivated viruses, which is remarkable considering the extraordinary diversity and abundance of viruses revealed by metagenomic analysis. Given the explosion of new enzymatic reagents derived from thermophilic prokaryotes over the past 40 years, those obtained from thermophilic viruses should be equally potent tools. This review discusses the still-limited state of the art regarding the functional biology and biotechnology of thermophilic viruses with a focus on DNA polymerases, ligases, endolysins, and coat proteins. Functional analysis of DNA polymerases and primase-polymerases from phages infecting Thermus, Aquificaceae, and Nitratiruptor has revealed new clades of enzymes with strong proofreading and reverse transcriptase capabilities. Thermophilic RNA ligase 1 homologs have been characterized from Rhodothermus and Thermus phages, with both commercialized for circularization of single-stranded templates. Endolysins from phages infecting Thermus, Meiothermus, and Geobacillus have shown high stability and unusually broad lytic activity against Gram-negative and Gram-positive bacteria, making them targets for commercialization as antimicrobials. Coat proteins from thermophilic viruses infecting Sulfolobales and Thermus strains have been characterized, with diverse potential applications as molecular shuttles. To gauge the scale of untapped resources for these proteins, we also document over 20,000 genes encoded by uncultivated viral genomes from high-temperature environments that encode DNA polymerase, ligase, endolysin, or coat protein domains., (© 2023 The Author(s).)

Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein-DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Each nucleated cell contains several hundreds of mitochondria, which are unique organelles in being under dual genome control. The mitochondria contain their own DNA, the mtDNA, but most of mitochondrial proteins are encoded by nuclear genes, including all the proteins required for replication, transcription, and repair of mtDNA. MtDNA replication is a continuous process that requires coordinated action of several enzymes that are part of the mtDNA replisome. It also requires constant supply of deoxyribonucleotide triphosphates(dNTPs) and interaction with other mitochondria for mixing and unifying the mitochondrial compartment. MtDNA maintenance defects are a growing list of disorders caused by defects in nuclear genes involved in different aspects of mtDNA replication. As a result of defects in these genes, mtDNA depletion and/or multiple mtDNA deletions develop in affected tissues resulting in variable manifestations that range from adult-onset mild disease to lethal presentation early in life., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

MtDNA mutations are one of the hallmarks of ageing and age-related diseases. It is well established that somatic point mutations accumulate in mtDNA of multiple organs and tissues with increasing age and heteroplasmy is universal in mammals. However, the origin of these mutations remains controversial. The long-lasting hypothesis stating that mtDNA mutations emanate from oxidative damage via a self-perpetuating mechanism has been extensively challenged in recent years. Contrary to this initial ascertainment, mtDNA appears to be well protected from action of reactive oxygen species (ROS) through robust protein coating and endomitochondrial microcompartmentalization. Extensive development of scrupulous high-throughput DNA sequencing methods suggests that an imperfect replication process, rather than oxidative lesions are the main sources of mtDNA point mutations, indicating that mtDNA polymerase γ (POLG) might be responsible for the majority of mtDNA mutagenic events. Here, we summarize the recent knowledge in prevention and defence of mtDNA oxidative lesions and discuss the plausible mechanisms of mtDNA point mutation generation and fixation., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)