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At least 1,351 records · Page 75

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a β-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8–4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.

59 BASIC BIOLOGICAL SCIENCES↗

A Review of Remote Welding and Nondestructive Examination Technologies for the DOE Standard Canister

The U.S. Department of Energy (DOE) manages a wide variety of spent nuclear fuel (SNF) that poses a unique management challenge. To help address this challenge, the DOE Standard Canister (DOESC), designed to remain sealed during handling, storage, transportation, and disposal, was conceptualized as a standardized containment vessel to accommodate DOE-managed SNF. Since 1999, several welding and examination processes have been independently developed for the DOESC’s closure welds. However, neither the DOESC nor these processes have been realized in an operational capacity. This review paper seeks to present and compare previously developed DOESC closure weld, nondestructive examination, and repair processes and technologies. Specific processes developed for the Idaho Spent Fuel Facility, in preparation for the Yucca Mountain geological repository, and the recent Road-Ready Demonstration Project are discussed. Further, specific focus is given to how different operating constraints and the American Society of Mechanical Engineers Boiler and Pressure Vessel Code (BPVC) have driven certain welding and nondestructive examination requirements. Historical DOESC welding and examination strategies are assessed against current regulatory and BPVC requirements. The comparison of welding processes, technologies, and DOESC designs presented in this review paper will inform further construction efforts for other commercial and DOE-managed SNF containments, including the DOESC.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

The Impact of Disruptions on the Economics of a Tokamak Power Plant

Tokamaks are often considered to be a leading candidate for near-term, cost-effective fusion energy, but these devices are susceptible to sudden loss of confinement events called disruptions. The threat of disruptions has garnered serious attention in research for the next generation of burning plasma experiments, such as ITER, but has received little treatment in economic studies of magnetic fusion energy. In this paper, we present a model for quantifying the effect of disruptions on the cost of electricity produced by a tokamak power plant (TPP). We outline the various ways disruptions increase costs and decrease revenues, introduce metrics to quantify these effects, and add them to a levelized cost of electricity (LCOE) model. Additionally, we identify several rate-limiting repair steps and introduce a classification system of disruption types based on the time to return to operations. We demonstrate how the LCOE model can be used to find the cost of electricity and the requirements for disruption handling of a TPP, and we further highlight where future research can have a strong impact in neutralizing the “showstopping” potential of disruptions.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

Cytogenetic follow-up studies on humans with internal and external exposure to ionizing radiation

Cells exposed to ionizing radiation have a wide spectrum of DNA lesions that include DNA single-strand breaks, DNA double-strand breaks (DSBs), oxidative base damage and DNA-protein crosslinks. Among them, DSB is the most critical lesion, which when mis-repaired leads to unstable and stable chromosome aberrations. Currently, chromosome aberration analysis is the preferred method for biological monitoring of radiation-exposed humans. Here, stable chromosome aberrations, such as inversions and balanced translocations, persist in the peripheral blood lymphocytes of radiation-exposed humans for several years and, therefore, are potentially useful tools to prognosticate the health risks of radiation exposure, particularly in the hematopoietic system. In this review, we summarize the cytogenetic follow-up studies performed by REAC/TS (Radiation Emergency Assistance Center/Training site, Oak Ridge, USA) on humans exposed to internal and external radiation. In the light of our observations as well as the data existing in the literature, this review attempts to highlight the importance of follow-up studies for predicting the extent of genomic instability and its impact on delayed health risks in radiation-exposed victims.

61 RADIATION PROTECTION AND DOSIMETRY↗

Effect of deposition rate on microstructure and mechanical properties of 17-4 PH stainless steel fabricated by laser engineered net shaping

Laser engineered net shaping (LENS) is an additive manufacturing technique for fabricating and repairing metallic components. However, the relationships between its process parameters and the resulting mechanical properties of alloys such as 17-4 precipitation-hardening (PH) stainless steel require further investigation to enable reliable application. This study examines the specific effect of LENS deposition rate on the microstructure and mechanical properties of 17-4 PH stainless steel. Specimens were fabricated at two different deposition rates (8.47 and 9.31 mm s−1), subjected to subsequent solution and H900 aging treatment, and then evaluated via tensile testing, hardness measurements, and microscopy. A higher deposition rate results in a finer grain structure but increased porosity, leading to greater ultimate tensile strength (∼1294 MPa) yet lower ductility (strain at failure ∼5.8%) compared to the slower deposition rate (∼1266 MPa, ∼9.0% strain). Hardness follows the same trend as tensile strength. Tensile fracture surfaces for both conditions exhibited a mixed mode of ductile dimples and brittle quasi-cleavage regions, with chromium/silicon oxide particles identified within dimples. Complementary finite element modeling indicates that small void fractions primarily reduce ductility by enhancing localized plasticity, with only a marginal decrease in tensile strength. These integrated experimental and numerical results elucidate the mechanical properties linked to deposition rate, providing insight into tailoring LENS processes to achieve desired properties of 17-4 PH stainless steel.

17-4 PH stainlesssteel↗

Model-based, in-situ, non-destructive qualification and certification of parts made by autonomous additive manufacturing

To address the significant productivity challenges associated with the qualification and certification (Q&C) tasks of additively manufactured (AM) parts, which have traditionally relied on rigorous post‐build inspection and testing, we propose an integrated framework that combines model‐based qualification and certification (MBQ&C) with autonomous additive manufacturing (AAM). MBQ&C employs high‐fidelity predictive models, developed within the Integrated Computational Materials Engineering (ICME) paradigm, to simulate process–structure–property–performance relationships for assessing a part’s fitness for use. Since predictive models are commonly machine learning (ML)-based or reduced-order surrogates of validated physics models, they run efficiently, enabling timely inference. In parallel, the self-driving AAM utilises ML-based adaptive, closed‐loop control strategies to avoid, mitigate, or repair defects and anomalies during fabrication, thereby increasing the likelihood of producing acceptable parts. A key feature of the combined AAM-MBQ&C framework is that predictive models explicitly incorporate defects or anomalies that persist after the build, using instance-specific data captured via in-situ sensing. This customisation enables a build‐specific assessment of fitness for use, rather than relying on nominal or generic parameters. Such individualised evaluation provides a robust basis for Q&C-related acceptance decisions relating to each build. Additionally, the rapid solution capabilities of ML or reduced-order models enable the determination of a part’s suitability for service shortly after build completion. As the framework matures, it has the potential to substantially reduce reliance on conventional point‐design approaches—such as time‐consuming post‐build computed tomography scanning and costly destructive testing. Thus, the AAM-MBQ&C framework represents a transformative, scalable strategy for quality assurance of AM components, as parts produced within a stable, validated, and certified envelope can be certified with reduced testing. Key benefits include: (1) significant gains in Q&C productivity through efficient, model-centric assessment; (2) performance-based classification of defects into critical and non-critical categories; (3) the ability to predict potential deviations in the performance of parts affected by real-time, adaptive process control interventions relative to those produced under a certified process, and (4) the enabling of virtual Q&C for service environments that are difficult, hazardous, or impractical to access or reproduce experimentally. Collectively, these capabilities strengthen the business case for AM, particularly for high‐consequence and mission‐critical applications. Finally, although this work focuses on powder-based AM, the proposed techniques could be extended to AM processes employing alternative feedstock forms.

Gunasegaram, Dayalan↗

Anticipating water distribution service outages from increasing temperatures

With projected temperature increases and extreme events due to climate change for many regions of the world, characterizing the impacts of these emerging hazards on water distribution systems is necessary to identify and prioritize adaptation strategies for ensuring reliability. To aid decision-making, new insights are needed into how water distribution system reliability to climate-driven heat will change, and the proactive maintenance strategies available to combat failures. To this end, we present the model Perses, a framework that joins a water distribution network hydraulic solver with reliability models of physical assets or components to estimate temperature increase-driven failures and resulting service outages in the long term. A theoretical case study is developed using Phoenix, Arizona temperature profiles, a city with extreme temperatures and a rapidly expanding infrastructure. By end-of-century under hotter futures there are projected to be 1%–5% more pump failures, 2%–5% more PVC pipe failures, and 3%–7% more iron pipe failures (RCP 4.5–8.5) than a baseline historical temperature profile. Service outages, which constitute inadequate pressure for domestic and commercial use are projected to increase by 16%–26% above the baseline under maximum temperature conditions. The exceedance of baseline failures, when compounded across a large metro region, reveals potential challenges for budgeting, management, and maintenance. An exploration of the mitigation potential of adaptation strategies shows that expedited repair times are capable of offsetting the additional outages from climate change, but will come with a cost.

54 ENVIRONMENTAL SCIENCES↗

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

59 BASIC BIOLOGICAL SCIENCES↗

Adding Tree Rings to North America's National Forest Inventories: An Essential Tool to Guide Drawdown of Atmospheric CO 2

Tree-ring time series provide long-term, annually resolved information on the growth of trees. When sampled in a systematic context, tree-ring data can be scaled to estimate the forest carbon capture and storage of landscapes, biomes, and—ultimately—the globe. A systematic effort to sample tree rings in national forest inventories would yield unprecedented temporal and spatial resolution of forest carbon dynamics and help resolve key scientific uncertainties, which we highlight in terms of evidence for forest greening (enhanced growth) versus browning (reduced growth, increased mortality). We describe jump-starting a tree-ring collection across the continent of North America, given the commitments of Canada, the United States, and Mexico to visit forest inventory plots, along with existing legacy collections. Failing to do so would be a missed opportunity to help chart an evidence-based path toward meeting national commitments to reduce net greenhouse gas emissions, urgently needed for climate stabilization and repair.

54 ENVIRONMENTAL SCIENCES↗

Syntrophic bacterial and host–microbe interactions in bacterial vaginosis

Bacterial vaginosis (BV) is a common, polymicrobial condition of the vaginal microbiota that is associated with symptoms such as malodor and excessive discharge, along with increased risk of various adverse sequelae. Host–bacteria and bacteria–bacteria interactions are thought to contribute to the condition, but many of these functions have yet to be elucidated. Using untargeted metaproteomics, we identified 1068 host and 1418 bacterial proteins in a set of cervicovaginal lavage samples collected from 20 participants with BV and 9 who were negative for the condition. We identified Dialister micraerophilus as a major producer of malodorous polyamines and identified a syntrophic interaction between this organism and Fannyhessea vaginae that leads to increased production of putrescine, a metabolite characteristic of BV. Although formate synthesis has not previously been noted in BV, we discovered diverse bacteria associated with the condition express pyruvate formate-lyase enzymes in vivo and confirm these organisms secrete formic acid in vitro. Sodium hypophosphite efficiently inhibited this function in multiple taxa. We also found that the fastidious organism Coriobacteriales bacterium DNF00809 can metabolize formic acid secreted by Gardnerella vaginalis, representing another syntrophic interaction. We noted an increased abundance of the host epithelial repair protein transglutaminase 3 in the metaproteomic data, which we confirmed by enzyme-linked immunosorbent assay. Other proteins identified in our samples implicate Finegoldia magna and Parvimonas micra in the production of malodorous trimethylamine. Some bacterial proteins identified represent novel targets for future therapeutics to disrupt BV communities and promote vaginal colonization by commensal lactobacilli.

bacterial vaginosis↗

Sulfide stress tolerance as a controller of methane production in temperate wetlands

Abstract Wetlands are a major source of methane emissions and contribute to the observed increase in atmospheric methane over the last 20 years. Methane production in wetlands is the final step of carbon decomposition performed by anaerobic archaea. Although hydrogen/carbon dioxide and acetate are the substrates most often attributed to methanogenesis, other substrates—such as methylated compounds—may additionally play important roles in driving methane production in wetland systems. Here we conducted mesocosm experiments combined with genome-resolved metatranscriptomics to investigate the impact of diverse methanogenic substrate amendment on methanogenesis in two high methane-emitting wetlands with distinct geochemistry, termed P7 and P8. Methanol amendment resulted in high methane production at both sites, whereas acetate and formate amendment only stimulated methanogenesis in P7 mesocosms, where aqueous sulfide concentrations were lower. In P7 sediments, formate amendment fueled acetogenic microbes that produced acetate, which was subsequently utilized by acetoclastic methanogens. In contrast to expression profiles in P7 mesocosms, active methylotrophic methanogen genomes from P8 showed increased expression of genes related to membrane remodeling and DNA damage repair, indicative of stress tolerance mechanisms to counter sulfide toxicity. Methylotrophic methanogenesis generates higher free energy yields than acetoclastic methanogenesis, which likely enables allocation of more energy toward stress responses. These findings contribute to the growing body of literature highlighting methylotrophic methanogenesis as an important methane production pathway in wetlands. By using less competitive substrates like methanol that provide greater energy yields, methylotrophic methanogens may invest in physiological strategies that provide competitive advantages across a range of environmental stresses.

Environmental Sciences & Ecology↗

Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP

Abstract Bacterial DNA methylation is involved in diverse cellular functions, including modulation of gene expression, DNA repair, and restriction–modification systems for defense against viruses and other foreign DNA. Restriction systems hinder efforts to engineer organisms to produce fuels and chemicals from waste and renewable feedstocks by degrading DNA during transformation. Methylome analysis allows identification of motifs within a bacterial chromosome that may be targeted by native restriction enzymes. Further expression of the corresponding methyltransferases in Escherichia coli allows plasmid DNA to be protected from restriction in the target organism, thereby drastically enhancing transformation efficiency. Nanopore sequencing can detect methylated bases, but software is needed to transform modified base coordinates into methylated motifs. Here, we develop MIJAMP (MIJAMP Is Just A MethylBED Parser), a software package that was developed to discover methylated motifs from the output of ONT’s Modkit or other data in the methylBED format. MIJAMP employs a human-driven refinement strategy that empirically validates all motifs against genome-wide methylation data, thus eliminating incorrect motifs. MIJAMP also reports methylation data on specific, user-defined motifs. Using MIJAMP, we determined the methylated motifs both in a control strain (wild-type E. coli) and in Synecococcus sp. strain PCC7002, laying the foundation for improved transformation in this organism. MIJAMP is available at https://code.ornl.gov/alexander-public/mijamp/. One Sentence Summary: Here we describe software written to discover DNA methylation motifs from nanopore sequencing data.

59 BASIC BIOLOGICAL SCIENCES↗

From quantitative trait loci towards mechanisms: Linkage Integration Hypothesis Testing (LIgHT) sheds light on the mechanisms of genetically modulated stress tolerance

The goal of this work is to assess the mechanistic bases of natural genetic variations in plant responses of photosynthesis to stress. To achieve this goal, we devised the Linkage Integration Hypothesis Testing (LIgHT) approach, comparing chromosomal locations of quantitative trait loci (QTLs) for multiple phenotypes to distinguish between hypothetical mechanisms. As a use case, we explored genetic variations in photosynthesis-related processes under chilling stress in recombinant inbred lines of cowpea ( Vigna unguiculata L. Walp.). We focused on photosynthesis-related parameters measurable in high throughput and indicative of proposed chilling responses, including the states of PSI and PSII, photoprotective non-photochemical quenching, PSII photodamage, and nyctinastic leaf movements (NLMs). The patterns of QTL linkages indicated that chilling stress tolerance is genetically controlled by avoiding PSII photodamage rather than PSI damage or NLMs. This model was validated in a separate experiment measuring the rates of PSII photodamage and repair. Additional linkages suggest that chilling-induced damage to PSII is controlled by the thylakoid proton motive force and redox state of PSII. This regulation appears to be modulated by thylakoid fatty acid composition, previously associated with the same genetic loci and now supported by broader mechanistic evidence. We propose that the LIgHT approach can be broadly applied to test mechanisms underlying genetic variations.

MultispeQ↗

CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

59 BASIC BIOLOGICAL SCIENCES↗

Improved genome editing by an engineered CRISPR-Cas12a

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis - and trans -DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast

Abstract Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

Carvalho Borges, Pâmela C. (ORCID:0000000244919874↗

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

59 BASIC BIOLOGICAL SCIENCES↗

Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ

Abstract The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.

Biochemistry & Molecular Biology↗