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The growth and development of metazoan organisms is dependent upon a co-ordinated programme of cellular proliferation and differentiation, from the initial formation of the zygote through to maintenance of mature organs in adult organisms. Early studies of proliferation of ex vivo cultures and unicellular eukaryotes described a cyclic nature of cell division characterised by periods of DNA synthesis (S-phase) and segregation of newly synthesized chromosomes (M-phase) interspersed by seeming inactivity, the gap phases, G1 and G2. We now know that G1 and G2 play critical roles in regulating the cell cycle, including monitoring of favourable environmental conditions to facilitate cell division, and ensuring genomic integrity prior to DNA replication and nuclear division. M-phase is usually followed by the physical separation of nascent daughters, termed cytokinesis. These phases where G1 leads to S phase, followed by G2 prior to M phase and the subsequent cytokinesis to produce two daughters, both identical in genomic composition and cellular morphology are what might be termed an archetypal cell division. Studies of development of many different organs in different species have demonstrated that this stereotypical cell cycle is often subverted to produce specific developmental outcomes, and examples from over 100 years of analysis of the development of Drosophila melanogaster have uncovered many different modes of cell division within this one species., (© 2024 The Author(s).)

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs., (© 2024 The Author(s).)

Meiotic recombination, a cornerstone of eukaryotic diversity and individual genetic identity, is essential for the creation of physical linkages between homologous chromosomes, facilitating their faithful segregation during meiosis I. This process requires that germ cells generate controlled DNA lesions within their own genome that are subsequently repaired in a specialised manner. Repair of these DNA breaks involves the modulation of existing homologous recombination repair pathways to generate crossovers between homologous chromosomes. Decades of genetic and cytological studies have identified a multitude of factors that are involved in meiotic recombination. Recent work has started to provide additional mechanistic insights into how these factors interact with one another, with DNA, and provide the molecular outcomes required for a successful meiosis. Here, we provide a review of the recent developments with a focus on protein structures and protein-protein interactions., (© 2024 The Author(s).)

Inosine, a purine nucleoside containing the hypoxanthine (HX) nucleobase, can form in DNA via hydrolytic deamination of adenine. Due to its structural similarity to guanine and the geometry of Watson-Crick base pairs, inosine can mispair with cytosine upon catalysis by DNA polymerases, leading to AT → GC mutations. Additionally, inosine plays an essential role in purine nucleotide biosynthesis, and inosine triphosphate is present in living cells. In a recent publication, Averill and Jung examined the possibility of polη catalyzed incorporation of deoxyinosine triphosphate (dITP) across dC and dT in a DNA template. They found that dITP can be incorporated across C or T, with the ratio of 13.7. X ray crystallography studies revealed that the mutagenic incorporation of dITP by human polη was affected by several factors including base pair geometry in the active site of the polymerase, tautomerization of nucleobases, and the interaction of the incoming dITP nucleotide with active site residues of polη. This study demonstrates that TLS incorporation of inosine monophosphate (IMP) into growing DNA chains contributes to its mutagenic potential in cells., (© 2023 The Author(s).)

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).)

Inosine is a key intermediate in de novo purine nucleotide biosynthesis in cells. Inosine is known to be mutagenic when it is present in DNA, in place of adenine via deamination, by facilitating the incorporation of dCTP exclusively, resulting in A:T to G:C mutation. The structural basis for the mutagenicity of inosine bypass has been reported in some DNA polymerases including human DNA polymerase eta (polη). However, the structural and biochemical basis for the mutagenic potential of the incorporation of deoxyinosine triphosphate (dITP) into DNA remains poorly understood. To gain insights into the mutagenic potential of the incorporation of inosine into DNA, we conducted structural and kinetic studies of human polη incorporating dITP across undamaged DNA template containing dC or dT. Polη incorporated dITP opposite dC 14-fold more efficiently than opposite dT, indicating that dITP incorporation by polη can be mutagenic unlike the bypass of inosine by polη, which incorporated dCTP almost exclusively opposite the templating inosine over dTTP (70:1). Polη-dC:dITP crystal structure showed that the incoming dITP formed Watson-Crick base pair along with wobble base pair via 4-imino-2-keto tautomer of cytosine diminishing the catalytic efficiency compared to dGTP incorporation across dC. In addition, the crystal structure of polη-dT:dITP revealed that dT and dITP formed Watson-Crick like base pair via 4-enol-2-keto tautomer of thymine, reinforced by wobble base pair via 4-keto-2-keto tautomer of thymine resulting in the increased mutagenicity of dITP incorporation (14:1 across dC and dT), which is 14-fold higher than dGTP incorporation by polη (190:1 across dC and dT)., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Chk1 is a member of the DNA damage response pathway, whose loss leads to replication stress and genome instability. Because of its protective role against lethal levels of DNA replication stress, Chk1 has been studied as a valuable and intriguing target for cancer therapy. However, one of the most prominent challenges with this strategy is development of resistance to Chk1 inhibitors, rendering the treatment ineffective. In their recent papers, Hunter and colleagues demonstrate multiple mechanisms by which Chk1 inhibitor resistance can arise in lymphomas. Specifically, this series of papers identify the relationship between dysfunction in NF-κB and the development of Chk1 inhibitor resistance through a loss of Chk1 activity in mouse models of lymphoma. They identify that cells lacking Chk1 activity can compensate for this loss through up-regulation of alternative pathways, such as PI3K/AKT. Finally, this work also identifies a novel role for Claspin, an important Chk1 activator, in female fertility and cancer development, furthering our understanding of how dysfunction in the Claspin/Chk1 signaling pathway affects disease states. These findings improve our understanding of drug resistance in cancer therapy, which has important implications for clinical use of Chk1 inhibitors., (© 2022 The Author(s).)

Claspin is an adaptor protein required for ATR-dependent phosphorylation of CHK1 during S-phase following DNA replication stress. Claspin expression is highly variable in cancer, with low levels frequently correlating with poor patient survival. To learn more about the biological consequences of reduced Claspin expression and its effects on tumorigenesis, we investigated mice with a heterozygous knockout of the Clspn gene. Claspin haploinsufficiency resulted in reduced female fertility and a maternally inherited defect in oocyte meiosis I cell cycle progression. Furthermore, aged Clspn+/- mice developed spontaneous lymphoid hyperplasia and increased susceptibility to non-alcoholic fatty liver disease. Importantly, we demonstrate a tumour suppressor role for Claspin. Reduced Claspin levels result in increased liver damage and tumourigenesis in the DEN model of hepatocellular carcinoma. These data reveal that Clspn haploinsufficiency has widespread unanticipated biological effects and establishes the importance of Claspin as a regulatory node controlling tumorigenesis and multiple disease aetiologies., (© 2022 The Author(s).)

The cell division cycle must be strictly regulated during both development and adult maintenance, and efficient and well-controlled DNA replication is a key event in the cell cycle. DNA replication origins are prepared in G1 phase of the cell cycle in a process known as origin licensing which is essential for DNA replication initiation in the subsequent S phase. Appropriate origin licensing includes: (1) Licensing enough origins at adequate origin licensing speed to complete licensing before G1 phase ends; (2) Licensing origins such that they are well-distributed on all chromosomes. Both aspects of licensing are critical for replication efficiency and accuracy. In this minireview, we will discuss recent advances in defining how origin licensing speed and distribution are critical to ensure DNA replication completion and genome stability., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function - deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria. Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction., (© 2021 The Author(s).)

The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended β-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended β-hairpin is involved in nucleotide incorporation on substrates with 5'-overhangs longer than 2 nt, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3'-5' exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating β-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.)

DNA polymerase δ (Polδ) is a highly processive essential replicative DNA polymerase. In humans, the Polδ holoenzyme consists of p125, p50, p68 and p12 subunits and recently, we showed that the p12 subunit exists as a dimer. Extensive biochemical studies suggest that all the subunits of Polδ interact with the processivity factor proliferating cell nuclear antigen (PCNA) to carry out a pivotal role in genomic DNA replication. While PCNA-interacting protein motif (PIP) motifs in p68, p50 and p12 have been mapped, same in p125, the catalytic subunit of the holoenzyme, remains elusive. Therefore, in the present study by using multiple approaches we have conclusively mapped a non-canonical PIP motif from residues 999VGGLLAFA1008 in p125, which binds to the inter-domain-connecting loop (IDCL) of PCNA with high affinity. Collectively, including previous studies, we conclude that similar to Saccharomyces cerevisiae Polδ, each of the human Polδ subunits possesses motif to interact with PCNA and significantly contributes toward the processive nature of this replicative DNA polymerase., (© 2020 The Author(s).)

Genetically identical cells frequently exhibit striking heterogeneity in various phenotypic traits such as their morphology, growth rate, or gene expression. Such non-genetic diversity can help clonal bacterial populations overcome transient environmental challenges without compromising genome stability, while genetic change is required for long-term heritable adaptation. At the heart of the balance between genome stability and plasticity are the DNA repair pathways that shield DNA from lesions and reverse errors arising from the imperfect DNA replication machinery. In principle, phenotypic heterogeneity in the expression and activity of DNA repair pathways can modulate mutation rates in single cells and thus be a source of heritable genetic diversity, effectively reversing the genotype-to-phenotype dogma. Long-standing evidence for mutation rate heterogeneity comes from genetics experiments on cell populations, which are now complemented by direct measurements on individual living cells. These measurements are increasingly performed using fluorescence microscopy with a temporal and spatial resolution that enables localising, tracking, and counting proteins with single-molecule sensitivity. In this review, we discuss which molecular processes lead to phenotypic heterogeneity in DNA repair and consider the potential consequences on genome stability and dynamics in bacteria. We further inspect these concepts in the context of DNA damage and mutation induced by antibiotics., (© 2020 The Author(s).)

The environmental Alphaproteobacterium Caulobacter crescentus is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S phase and a swarmer cell that stays in the G1 phase until it differentiates into a stalked cell. Its genome consists in a single circular chromosome whose replication is tightly regulated so that it happens only in stalked cells and only once per cell cycle. Imbalances in chromosomal copy numbers are the most often highly deleterious, if not lethal. This review highlights recent discoveries on pathways that control chromosome replication when Caulobacter is exposed to optimal or less optimal growth conditions. Most of these pathways target two proteins that bind directly onto the chromosomal origin: the highly conserved DnaA initiator of DNA replication and the CtrA response regulator that is found in most Alphaproteobacteria The concerted inactivation and proteolysis of CtrA during the swarmer-to-stalked cell transition license cells to enter S phase, while a replisome-associated Regulated Inactivation and proteolysis of DnaA (RIDA) process ensures that initiation starts only once per cell cycle. When Caulobacter is stressed, it turns on control systems that delay the G1-to-S phase transition or the elongation of DNA replication, most probably increasing its fitness and adaptation capacities., (© 2019 The Author(s).)

RecJ proteins belong to the DHH superfamily of phosphoesterases that has members in all three domains of life. In bacteria, the archetypal RecJ is a 5' → 3' ssDNA exonuclease that functions in homologous recombination, base excision repair and mismatch repair, while in eukaryotes, the RecJ-like protein Cdc45 (which has lost its nuclease activity) is a key component of the CMG (Cdc45-MCM-GINS) complex, the replicative DNA helicase that unwinds double-stranded DNA at the replication fork. In archaea, database searching identifies genes encoding one or more RecJ family proteins in almost all sequenced genomes. Biochemical analysis has confirmed that some but not all of these proteins are components of archaeal CMG complexes and has revealed a surprising diversity in mode of action and substrate preference. In addition to this, some archaea encode catalytically inactive RecJ-like proteins, and others a mix of active and inactive proteins, with the inactive proteins being confined to structural roles only. Here, I summarise current knowledge of the structure and function of the archaeal RecJ-like proteins, focusing on similarities and differences between proteins from different archaeal species, between proteins within species and between the archaeal proteins and their bacterial and eukaryotic relatives. Models for RecJ-like function are described and key areas for further study highlighted., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Archaeal DNA polymerases have long been studied due to their superior properties for DNA amplification in the polymerase chain reaction and DNA sequencing technologies. However, a full comprehension of their functions, recruitment and regulation as part of the replisome during genome replication and DNA repair lags behind well-established bacterial and eukaryotic model systems. The archaea are evolutionarily very broad, but many studies in the major model systems of both Crenarchaeota and Euryarchaeota are starting to yield significant increases in understanding of the functions of DNA polymerases in the respective phyla. Recent advances in biochemical approaches and in archaeal genetic models allowing knockout and epitope tagging have led to significant increases in our understanding, including DNA polymerase roles in Okazaki fragment maturation on the lagging strand, towards reconstitution of the replisome itself. Furthermore, poorly characterised DNA polymerase paralogues are finding roles in DNA repair and CRISPR immunity. This review attempts to provide a current update on the roles of archaeal DNA polymerases in both DNA replication and repair, addressing significant questions that remain for this field., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

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.)

Helicases are molecular motors that play central roles in nucleic acid metabolism. Mutations in genes encoding DNA helicases of the RecQ and iron-sulfur (Fe-S) helicase families are linked to hereditary disorders characterized by chromosomal instabilities, highlighting the importance of these enzymes. Moreover, mono-allelic RecQ and Fe-S helicase mutations are associated with a broad spectrum of cancers. This review will discuss and contrast the specialized molecular functions and biological roles of RecQ and Fe-S helicases in DNA repair, the replication stress response, and the regulation of gene expression, laying a foundation for continued research in these important areas of study., (© 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.)

PrimPol, (prim ase- pol ymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered in vitro , we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process., (© 2017 The Author(s).)