SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

DNA polymerase ß outperforms DNA polymerase γ in key mitochondrial base excision repair activities.

DNA polymerase beta (POLß), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLß and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLß is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLß has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLß are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLß may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.

Current and emerging roles of Cockayne syndrome group B (CSB) protein.

Cockayne syndrome (CS) is a segmental premature aging syndrome caused primarily by defects in the CSA or CSB genes. In addition to premature aging, CS patients typically exhibit microcephaly, progressive mental and sensorial retardation and cutaneous photosensitivity. Defects in the CSB gene were initially thought to primarily impair transcription-coupled nucleotide excision repair (TC-NER), predicting a relatively consistent phenotype among CS patients. In contrast, the phenotypes of CS patients are pleiotropic and variable. The latter is consistent with recent work that implicates CSB in multiple cellular systems and pathways, including DNA base excision repair, interstrand cross-link repair, transcription, chromatin remodeling, RNAPII processing, nucleolin regulation, rDNA transcription, redox homeostasis, and mitochondrial function. The discovery of additional functions for CSB could potentially explain the many clinical phenotypes of CSB patients. This review focuses on the diverse roles played by CSB in cellular pathways that enhance genome stability, providing insight into the molecular features of this complex premature aging disease.

Skin Abnormalities in Disorders with DNA Repair Defects, Premature Aging, and Mitochondrial Dysfunction.

Defects in DNA repair pathways and alterations of mitochondrial energy metabolism have been reported in multiple skin disorders. More than 10% of patients with primary mitochondrial dysfunction exhibit dermatological features including rashes and hair and pigmentation abnormalities. Accumulation of oxidative DNA damage and dysfunctional mitochondria affect cellular homeostasis leading to increased apoptosis. Emerging evidence demonstrates that genetic disorders of premature aging that alter DNA repair pathways and cause mitochondrial dysfunction, such as Rothmund-Thomson syndrome, Werner syndrome, and Cockayne syndrome, also exhibit skin disease. This article summarizes recent advances in the research pertaining to these syndromes and molecular mechanisms underlying their skin pathologies.

DNA damage and mitochondria in cancer and aging.

Age and DNA repair deficiencies are strong risk factors for developing cancer. This is reflected in the comorbidity of cancer with premature aging diseases associated with DNA damage repair deficiencies. Recent research has suggested that DNA damage accumulation, telomere dysfunction and the accompanying mitochondrial dysfunction exacerbate the aging process and may increase the risk of cancer development. Thus, an area of interest in both cancer and aging research is the elucidation of the dynamic crosstalk between the nucleus and the mitochondria. In this review, we discuss current research on aging and cancer with specific focus on the role of mitochondrial dysfunction in cancer and aging as well as how nuclear to mitochondrial DNA damage signaling may be a driving factor in the increased cancer incidence with aging. We suggest that therapeutic interventions aimed at the induction of autophagy and mediation of nuclear to mitochondrial signaling may provide a mechanism for healthier aging and reduced tumorigenesis.

Enhanced mitochondrial DNA repair of the common disease-associated variant, Ser326Cys, of hOGG1 through small molecule intervention.

The common oxidatively generated lesion, 8-oxo-7,8-dihydroguanine (8-oxoGua), is removed from DNA by base excision repair. The glycosylase primarily charged with recognition and removal of this lesion is 8-oxoGuaDNA glycosylase 1 (OGG1). When left unrepaired, 8-oxodG alters transcription and is mutagenic. Individuals homozygous for the less active OGG1 allele, Ser326Cys, have increased risk of several cancers. Here, small molecule enhancers of OGG1 were identified and tested for their ability to stimulate DNA repair and protect cells from the environmental hazard paraquat (PQ). PQ-induced mtDNA damage was inversely proportional to the levels of OGG1 expression whereas stimulation of OGG1, in some cases, entirely abolished its cellular effects. The PQ-mediated decline of mitochondrial membrane potential or nuclear condensation were prevented by the OGG1 activators. In addition, in Ogg1-/- mouse embryonic fibroblasts complemented with hOGG1S326C, there was increased cellular and mitochondrial reactive oxygen species compared to their wild type counterparts. Mitochondrial extracts from cells expressing hOGG1S326C were deficient in mitochondrial 8-oxodG incision activity, which was rescued by the OGG1 activators. These data demonstrate that small molecules can stimulate OGG1 activity with consequent cellular protection. Thus, OGG1-activating compounds may be useful in select humans to mitigate the deleterious effects of environmental oxidants and mutagens.

NAD+ supplementation normalizes key Alzheimer's features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency.

Emerging findings suggest that compromised cellular bioenergetics and DNA repair contribute to the pathogenesis of Alzheimer's disease (AD), but their role in disease-defining pathology is unclear. We developed a DNA repair-deficient 3xTgAD/Polß+/- mouse that exacerbates major features of human AD including phosphorylated Tau (pTau) pathologies, synaptic dysfunction, neuronal death, and cognitive impairment. Here we report that 3xTgAD/Polß+/- mice have a reduced cerebral NAD+/NADH ratio indicating impaired cerebral energy metabolism, which is normalized by nicotinamide riboside (NR) treatment. NR lessened pTau pathology in both 3xTgAD and 3xTgAD/Polß+/- mice but had no impact on amyloid ß peptide (Aß) accumulation. NR-treated 3xTgAD/Polß+/- mice exhibited reduced DNA damage, neuroinflammation, and apoptosis of hippocampal neurons and increased activity of SIRT3 in the brain. NR improved cognitive function in multiple behavioral tests and restored hippocampal synaptic plasticity in 3xTgAD mice and 3xTgAD/Polß+/- mice. In general, the deficits between genotypes and the benefits of NR were greater in 3xTgAD/Polß+/- mice than in 3xTgAD mice. Our findings suggest a pivotal role for cellular NAD+ depletion upstream of neuroinflammation, pTau, DNA damage, synaptic dysfunction, and neuronal degeneration in AD. Interventions that bolster neuronal NAD+ levels therefore have therapeutic potential for AD.

DNA polymerase ß decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition, patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase-ß (Polß) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild-type and 3xTgAD mice with normal or reduced Polß levels. Compared to wild-type control mice, Polß heterozygous (Polß+/- ), and 3xTgAD mice, 3xTgAD/Polß+/- mice exhibited impaired performance in a buried food test of olfaction. Polß deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Polß+/- and 3xTgAD mice, and by over 60% in the 3xTgAD/Polß+/- mice compared to wild-type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polß+/- mice compared to 3xTgAD mice. Levels of amyloid ß-peptide (Aß) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polß+/- mice, and cultured Polß-deficient neurons exhibited increased vulnerability to Aß-induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD.

Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites.

Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, ß, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol ß did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide.

DNA polymerase ß deficiency leads to neurodegeneration and exacerbates Alzheimer disease phenotypes.

We explore the role of DNA damage processing in the progression of cognitive decline by creating a new mouse model. The new model is a cross of a common Alzheimer's disease (AD) mouse (3xTgAD), with a mouse that is heterozygous for the critical DNA base excision repair enzyme, DNA polymerase ß. A reduction of this enzyme causes neurodegeneration and aggravates the AD features of the 3xTgAD mouse, inducing neuronal dysfunction, cell death and impairing memory and synaptic plasticity. Transcriptional profiling revealed remarkable similarities in gene expression alterations in brain tissue of human AD patients and 3xTg/Polß(+/-) mice including abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in base excision repair capacity can render the brain more vulnerable to AD-related molecular and cellular alterations.

Mature microsatellites: mechanisms underlying dinucleotide microsatellite mutational biases in human cells.

Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (≥10 units) are present within exonic sequences of >350 genes, resulting in vulnerability to cellular genetic integrity. Mature dinucleotide mutagenesis was examined experimentally using ex vivo and in vitro approaches. We observe an expansion bias for dinucleotide microsatellites up to 20 units in length in somatic human cells, in agreement with previous computational analyses of germ-line biases. Using purified DNA polymerases and human cell lines deficient for mismatch repair (MMR), we show that the expansion bias is caused by functional MMR and is not due to DNA polymerase error biases. Specifically, we observe that the MutSα and MutLα complexes protect against expansion mutations. Our data support a model wherein different MMR complexes shift the balance of mutations toward deletion or expansion. Finally, we show that replication fork progression is stalled within long dinucleotides, suggesting that mutational mechanisms within long repeats may be distinct from shorter lengths, depending on the biochemistry of fork resolution. Our work combines computational and experimental approaches to explain the complex mutational behavior of dinucleotide microsatellites in humans.

DNA polymerase kappa microsatellite synthesis: two distinct mechanisms of slippage-mediated errors.

Microsatellite tandem repeats are frequent sites of strand slippage mutagenesis in the human genome. Microsatellite mutations often occur as insertion/deletion of a repeat motif (unit-based indels), and increase in frequency with increasing repeat length after a threshold is reached. We recently demonstrated that DNA polymerase κ (Pol κ) produces fewer unit-based indel errors within dinucleotide microsatellites than does polymerase δ. Here, we examined human Pol κ's error profile within microsatellite alleles of varying sequence composition and length, using an in vitro HSV-tk gap-filling assay. We observed that Pol κ displays relatively accurate synthesis for unit-based indels, using di- and tetranucleotide repeat templates longer than the threshold length. We observed an abrupt increase in the unit-based indel frequency when the total microsatellite length exceeds 28 nucleotides, suggesting that extended Pol κ protein-DNA interactions enhance fidelity of the enzyme when synthesizing these microsatellite alleles. In contrast, Pol κ is error-prone within the HSV-tk coding sequence, producing frequent single-base errors in a manner that is highly biased with regard to sequence context. Single-nucleotide errors are also created by Pol κ within di- and tetranucleotide repeats, independently of the microsatellite allele length and at a frequency per nucleotide similar to the frequency of single base errors within the coding sequence. These single-base errors represent the mutational signature of Pol κ, and we propose them a mechanism independent of homology-stabilized slippage. Pol κ's dual fidelity nature provides a unique research tool to explore the distinct mechanisms of slippage-mediated mutagenesis.

RRD1, a component of the TORC1 signalling pathway, affects anaesthetic response in Saccharomyces cerevisiae.

The molecular mechanisms of action of volatile anaesthetics remain unknown despite clinical use for over 150 years. While many effects of these agents have been characterized, clear insight into how these effects relate to the physiological state of anaesthesia has not been established. Volatile anaesthetics arrest cell division in Saccharomyces cerevisiae in a manner that parallels the anaesthetic actions of these drugs in mammals. To gain additional insight into the cellular activities of these drugs, we isolated genes that, when present on multi-copy plasmids, render S. cerevisiae resistant to the volatile anaesthetic isoflurane. One of these genes, RRD1, encodes a subunit of the Tap42p-Sit4p-Rrd1p phosphatase complex that functions in the target of rapamycin complex 1 (TORC1) signalling pathway. In addition, we show that mutations in two other genes encoding components of the TORC1 pathway, GLN3 and URE2, also affect yeast anaesthetic response. These findings suggest that TORC1-mediated signalling is involved in cellular response to volatile anaesthetics in S. cerevisiae.

Inhibition of translation initiation by volatile anesthetics involves nutrient-sensitive GCN-independent and -dependent processes in yeast.

Volatile anesthetics including isoflurane affect all cells examined, but their mechanisms of action remain unknown. To investigate the cellular basis of anesthetic action, we are studying Saccharomyces cerevisiae mutants altered in their response to anesthetics. The zzz3-1 mutation renders yeast isoflurane resistant and is an allele of GCN3. Gcn3p functions in the evolutionarily conserved general amino acid control (GCN) pathway that regulates protein synthesis and gene expression in response to nutrient availability through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). Hyperphosphorylation of eIF2alpha inhibits translation initiation during amino acid starvation. Isoflurane rapidly (in <15 min) inhibits yeast cell division and amino acid uptake. Unexpectedly, phosphorylation of eIF2alpha decreased dramatically upon initial exposure although hyperphosphorylation occurred later. Translation initiation was inhibited by isoflurane even when eIF2alpha phosphorylation decreased and this inhibition was GCN-independent. Maintenance of inhibition required GCN-dependent hyperphosphorylation of eIF2alpha. Thus, two nutrient-sensitive stages displaying unique features promote isoflurane-induced inhibition of translation initiation. The rapid phase is GCN-independent and apparently has not been recognized previously. The maintenance phase is GCN-dependent and requires inhibition of general translation imparted by enhanced eIF2alpha phosphorylation. Surprisingly, as shown here, the transcription activator Gcn4p does not affect anesthetic response.