Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Dynamics of Cas10 Govern Discrimination between Self and Non-self in Type III CRISPR-Cas Immunity.

Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity.

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

A Tour de Force on the Double Helix: Exploiting DNA Mechanics To Study DNA-Based Molecular Machines.

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell's operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale.

Single-molecule fluorescence microscopy is uniquely suited for detecting transient molecular recognition events, yet achieving the time resolution and statistics needed to realize this potential has proven challenging. Here we present a single-molecule imaging and analysis platform using scientific complementary metal-oxide semiconductor (sCMOS) detectors that enables imaging of 15,000 individual molecules simultaneously at millisecond rates. This system enabled the detection of previously obscured processes relevant to the fidelity mechanism in protein synthesis.

Direct evidence of an elongation factor-Tu/Ts·GTP·Aminoacyl-tRNA quaternary complex.

During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.

Ultra-stable organic fluorophores for single-molecule research.

Fluorescence provides a mechanism for achieving contrast in biological imaging that enables investigations of molecular structure, dynamics, and function at high spatial and temporal resolution. Small-molecule organic fluorophores have proven essential for such efforts and are widely used in advanced applications such as single-molecule and super-resolution microscopy. Yet, organic fluorophores, like all fluorescent species, exhibit instabilities in their emission characteristics, including blinking and photobleaching that limit their utility and performance. Here, we review the photophysics and photochemistry of organic fluorophores as they pertain to mitigating such instabilities, with a specific focus on the development of stabilized fluorophores through derivatization. Self-healing organic fluorophores, wherein the triplet state is intramolecularly quenched by a covalently attached protective agent, exhibit markedly improved photostabilities. We discuss the potential for further enhancements towards the goal of developing "ultra-stable" fluorophores spanning the visible spectrum and how such fluorophores are likely to impact the future of single-molecule research.

Cost-effectiveness of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) testing and isolation strategies in nursing homes.

OBJECTIVE: Nursing home residents may be particularly vulnerable to coronavirus disease 2019 (COVID-19). Therefore, a question is when and how often nursing homes should test staff for COVID-19 and how this may change as severe acute respiratory coronavirus virus 2 (SARS-CoV-2) evolves. DESIGN: We developed an agent-based model representing a typical nursing home, COVID-19 spread, and its health and economic outcomes to determine the clinical and economic value of various screening and isolation strategies and how it may change under various circumstances. RESULTS: Under winter 2023-2024 SARS-CoV-2 omicron variant conditions, symptom-based antigen testing averted 4.5 COVID-19 cases compared to no testing, saving $191 in direct medical costs. Testing implementation costs far outweighed these savings, resulting in net costs of $990 from the Centers for Medicare & Medicaid Services perspective, $1,545 from the third-party payer perspective, and $57,155 from the societal perspective. Testing did not return sufficient positive health effects to make it cost-effective [$50,000 per quality-adjusted life-year (QALY) threshold], but it exceeded this threshold in ≥59% of simulation trials. Testing remained cost-ineffective when routinely testing staff and varying face mask compliance, vaccine efficacy, and booster coverage. However, all antigen testing strategies became cost-effective (≤$31,906 per QALY) or cost saving (saving ≤$18,372) when the severe outcome risk was ≥3 times higher than that of current omicron variants. CONCLUSIONS: SARS-CoV-2 testing costs outweighed benefits under winter 2023-2024 conditions; however, testing became cost-effective with increasingly severe clinical outcomes. Cost-effectiveness can change as the epidemic evolves because it depends on clinical severity and other intervention use. Thus, nursing home administrators and policy makers should monitor and evaluate viral virulence and other interventions over time.

How the Timing of Annual COVID-19 Vaccination of Nursing Home Residents and Staff Affects Its Value.

OBJECTIVES: To evaluate the epidemiologic, clinical, and economic value of an annual nursing home (NH) COVID-19 vaccine campaign and the impact of when vaccination starts. DESIGN: Agent-based model representing a typical NH. SETTING AND PARTICIPANTS: NH residents and staff. METHODS: We used the model representing an NH with 100 residents, its staff, their interactions, COVID-19 spread, and its health and economic outcomes to evaluate the epidemiologic, clinical, and economic value of varying schedules of annual COVID-19 vaccine campaigns. RESULTS: Across a range of scenarios with a 60% vaccine efficacy that wanes starting 4 months after protection onset, vaccination was cost saving or cost-effective when initiated in the late summer or early fall. Annual vaccination averted 102 to 105 COVID-19 cases when 30-day vaccination campaigns began between July and October (varying with vaccination start), decreasing to 97 and 85 cases when starting in November and December, respectively. Starting vaccination between July and December saved $3340 to $4363 and $64,375 to $77,548 from the Centers for Medicare & Medicaid Services and societal perspectives, respectively (varying with vaccination start). Vaccination's value did not change when varying the COVID-19 peak between December and February. The ideal vaccine campaign timing was not affected by reducing COVID-19 levels in the community, or varying transmission probability, preexisting immunity, or COVID-19 severity. However, if vaccine efficacy wanes more quickly (over 1 month), earlier vaccination in July resulted in more cases compared with vaccinating later in October. CONCLUSIONS AND IMPLICATIONS: Annual vaccination of NH staff and residents averted the most cases when initiated in the late summer through early fall, at least 2 months before the COVID-19 winter peak but remained cost saving or cost-effective when it starts in the same month as the peak. This supports tethering COVID vaccination to seasonal influenza campaigns (typically in September-October) for providing protection against SARS-CoV-2 winter surges in NHs.

Modeling Nursing Home Harms From COVID-19 Staff Furlough Policies.

Importance: Current guidance to furlough health care staff with mild COVID-19 illness may prevent the spread of COVID-19 but may worsen nursing home staffing shortages as well as health outcomes that are unrelated to COVID-19. Objective: To compare COVID-19-related with non-COVID-19-related harms associated with allowing staff who are mildly ill with COVID-19 to work while masked. Design, Setting, and Participants: This modeling study, conducted from November 2023 to June 2024, used an agent-based model representing a 100-bed nursing home and its residents, staff, and their interactions; care tasks; and resident and staff health outcomes to simulate the impact of different COVID-19 furlough policies over 1 postpandemic year. Exposures: Simulating increasing proportions of staff who are mildly ill and are allowed to work while wearing N95 respirators under various vaccination coverage, SARS-CoV-2 transmissibility and severity, and masking adherence. Main Outcomes and Measures: The main outcomes were staff and resident COVID-19 cases, staff furlough days, missed care tasks, nursing home resident hospitalizations (related and unrelated to COVID-19), deaths, and costs. Results: In the absence of SARS-CoV-2 infection in the study's 100-bed agent-based model, nursing home understaffing resulted in an annual mean (SD) 93.7 (0.7) missed care tasks daily (22.1%), 38.0 (7.6) resident hospitalizations (5.2%), 4.6 (2.2) deaths (0.6%), and 39.7 (19.8) quality-adjusted life years lost from non-COVID-19-related harms, costing $1 071 950 ($217 200) from the Centers for Medicare & Medicaid Services (CMS) perspective and $1 112 800 ($225 450) from the societal perspective. Under the SARS-CoV-2 Omicron variant conditions from 2023 to 2024, furloughing all staff who tested positive for SARS-CoV-2 was associated with a mean (SD) 326.5 (69.1) annual furlough days and 649.5 (95% CI, 593.4-705.6) additional missed care tasks, resulting in 4.3 (95% CI, 2.9-5.9) non-COVID-19-related resident hospitalizations and 0.7 (95% CI, 0.2-1.1) deaths, costing an additional $247 090 (95% CI, $203 160-$291 020) from the CMS perspective and $405 250 (95% CI, $358 550-$451 950) from the societal perspective. Allowing 75% of staff who were mildly ill to work while masked was associated with 5 additional staff and 5 additional resident COVID-19 cases without added COVID-19-related hospitalizations but mitigated staffing shortages, with 475.9 additional care tasks being performed annually, 3.5 fewer non-COVID-19-related hospitalizations, and 0.4 fewer non-COVID-19-related deaths. Allowing staff who were mildly ill to work ultimately saved an annual mean $85 470 (95% CI, $41 210-$129 730) from the CMS perspective and $134 450 (95% CI, $86 370-$182 540) from the societal perspective. These results were robust to increased vaccination coverage, increased nursing home transmission, increased importation of COVID-19 from the community, and failure to mask while working ill. Conclusion and Relevance: In this modeling study of staff COVID-19 furlough policies, allowing nursing home staff to work with mild COVID-19 illness was associated with fewer resident harms from staffing shortages and missed care tasks than harms from increased COVID-19 transmission, ultimately saving substantial direct medical and societal costs.

Association of anemia with worsened activities of daily living and health-related quality of life scores derived from the Minimum Data Set in long-term care residents.

BACKGROUND: Among long-term care (LTC) residents, we explored the association between anemia status and hemoglobin (Hb) level with Activities of Daily Living (ADL) functioning and health-related quality of life (HRQOL). METHODS: Data were derived from the AnalytiCare database, containing laboratory and Minimum Data Set (MDS) reports for 27 LTC facilities in Colorado. Study timeframe was 1/1/07-9/15/08. Patients were selected based on: residence in LTC >90 days, Hb and serum creatinine value within 90 days of the earliest non-admission (index) MDS. From the index MDS, the method of 1) Carpenter et al. [BMC Geriatrics 6:7(2006)] was used to derive a summary measure of ADL performance (the MDS-ADL score) and 2) Wodchis et al. [IJTAHC 19:3(2003)] was used to assign HRQOL scores (MDS items were mapped to the Health Utilities Index Mark 2 (HUI2) scoring function to create the MDS-HSI score). Anemia was defined as Hb <12 g/dL females and <13 g/dL males. Adjusted linear regression was used to evaluate the independent association of anemia and hemoglobin level on MDS-ADL and MDS-HSI scores. RESULTS: 838 residents met all inclusion criteria; 46% of residents were anemic. Mean (SD) MDS-ADL score was 14.9 (7.5) [0-28 scale, where higher score indicates worse functioning]. In the adjusted model, anemia was associated with a significantly worse MDS-ADL score (+1.62 points, P=.001). Residents with Hb levels 10 to <11 g/dL had significantly worse ADL score (+2.06 points, P=.005) than the >13 g/dL reference. The mean MDS-HSI score was 0.431 (0.169) [range, where 0=dead to 1=perfect health]. Compared with non-anemic residents, in this adjusted model, residents with anemia had significantly worse MDS-HSI scores (-0.034 points, P=.005). Residents with hemoglobin levels <10 g/dL had significantly worse MDS-HSI scores (-0.058 points, P=.016) than the >13 g/dL reference. CONCLUSIONS: After adjusting for several covariates, LTC residents with anemia, and many of those with moderate to severe declines in Hb level, had significantly poorer outcomes in both ADL functioning and HRQOL. The association between Hb level and the HRQOL measure of MDS-HSI appears to be largely explained by the mobility domain of the HRQOL measure.

The prevalence and recognition of chronic kidney disease and anemia in long-term care residents.

OBJECTIVE: To evaluate the prevalence of chronic kidney disease (CKD) and anemia in the long-term care facility, the rate of recognition of these conditions, and the specific interventions used to treat anemia. DESIGN: Retrospective cross-sectional analysis. SETTING: Twenty-seven long-term care facilities in Colorado. PATIENTS, PARTICIPANTS: Had > 90-day residency in the long-term care facility; had index serum creatinine and hemoglobin (Hb) values ± 90 days of the earliest (index) Minimum Data Set (MDS). Data were derived from the AnalytiCare(sm) database (January 1, 2007-September 15, 2008) containing laboratory results, MDS reports, and pharmacy fills. Residents with laboratory-defined CKD had estimated glomerular filtration rates < 60 mL/min/1.73 m(2). Those with laboratory-defined anemia had < 12 g/dL Hb females, < 13 g/dL Hb males. MDS reports indicated recognition of CKD and anemia. Prescription records identified anemia-related pharmacotherapy for anemic residents. MAIN OUTCOME MEASUREMENTS: Prevalence rates of laboratory-defined CKD and anemia, recognition rates of anemia and CKD, and rates of use of specific anemia pharmacotherapies. RESULTS: For 838 eligible residents, laboratory findings showed a prevalence rate of 43% for CKD and 46% for anemia. Only 2.8% and 14.6% of residents with laboratory defined CKD had CKD recognized on the index, or any index or postindex MDS, respectively. Anemia recognition rates were 9.6% and 39.9%, respectively. No single anemia prescription therapy class (erythropoiesis stimulating agents, iron, vitamin B(12), or folic acid) was used for more than 10% of all residents with laboratory- or MDS-defined anemia. CONCLUSION: For CKD and anemia, the lack of concordance between laboratory- and MDS-identified disease should alert health care professionals of potential under-recognition within the long-term care facility.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence.

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes.

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.

Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses.

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

High-resolution structure of the Escherichia coli ribosome.

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation have remained poorly understood. Moreover, the functions of modifications to ribosomal RNA and ribosomal proteins have also been unclear. Here we present the structure of the Escherichia coli 70S ribosome at 2.4-Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and it suggests how solvation contributes to ribosome integrity and function as well as how the conformation of ribosomal protein uS12 aids in mRNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including post-transcriptional and post-translational modifications, and should serve as a basis for future antibiotic development.

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions.

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.