Molecular basis for transposase activation by a dedicated AAA+ ATPase.

Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1-3. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators-which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements-can remodel their substrate DNA and cognate transposases to promote function.

Structure of Escherichia coli exonuclease VII.

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped ß-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

Using energy to go downhill-a genoprotective role for ATPase activity in DNA topoisomerase II.

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction cycle. Curiously, most type II topoisomerases (topos II, IV and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase IIß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss elevates the enzyme's propensity for DNA damage. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in ATPase-less enzymes, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavage in vitro, as well as in vivo. By contrast, aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are present. Our findings are consistent with the proposal that type II topoisomerases acquired ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

Chromatinization Modulates Topoisomerase II Processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress in vivo. While cellular processes constantly create different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II). We observed that topo II was at least ~ 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a ~100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive (~ 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.

Chromatinization modulates topoisomerase II processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin's predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

Supplemental findings of the 2021 National Blood Collection and Utilization Survey.

BACKGROUND: The Department of Health and Human Services' National Blood Collection and Utilization Survey (NBCUS) has been conducted biennially since 1997. Data are used to estimate national blood collection and use. Supplemental data from the 2021 NBCUS not presented elsewhere are presented here. METHODS: Data on survey participation, donor characteristics, blood component cost, transfusion-associated adverse reactions, and implementation of blood safety measures, including pathogen-reduction of platelets, during 2021, were analyzed. Comparisons are made to 2019 survey data where available (2013-2019 for survey participation). RESULTS: During 2021, there were 11,507,000 successful blood donations in the United States, a 4.8% increase from 2019. Persons aged 45-64 years accounted for 42% of all successful blood donations. Donations by persons aged 65 years and older increased by 40.7%, while donations among minorities and donors aged <25 years decreased. From 2019 to 2021, the median price hospitals paid per unit of leukoreduced red blood cells, leukoreduced and pathogen-reduced apheresis platelets, and fresh frozen plasma increased. The largest increase in price per unit of blood component in 2021 was for leukoreduced apheresis platelets, which increased by ~$51. Between 2019 and 2021, the proportion of transfusing facilities reporting use of pathogen-reduced platelets increased, from 13% to 60%. Transfusion-related adverse reactions declined slightly between 2019 and 2021, although the rate of transfusion-transmitted bacterial infections remained unchanged. CONCLUSION: During 2021, blood donations increased nationally, although donations from those aged <25 years and minorities declined. The prices hospitals paid for most blood products increased, as did the use of pathogen-reduced platelets.

Using energy to go downhill - a genoprotective role for ATPase activity in DNA topoisomerase II.

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction. Curiously, most type II topoisomerases (topos II, IV, and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase II ß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss leads to increased DNA nicking and double strand break formation by the enzyme. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in the absence of the ATPase regions, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleava in ge vitro , as well as in vivo . By contrast, the aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are restored. Our findings are consistent with the proposal that type II topoisomerases acquired an ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

Naturally mutagenic sequence diversity in a human type II topoisomerase.

Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2ß) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in TOP2B sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing TOP2B alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2ß to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.

Impact of the COVID-19 pandemic on blood donation and transfusions in the United States in 2020.

INTRODUCTION: Reports have suggested the COVID-19 pandemic resulted in blood donation shortages and adverse impacts on the blood supply. Using data from the National Blood Collection and Utilization Survey (NBCUS), we quantified the pandemic's impact on red blood cell (RBC) and apheresis platelet collections and transfusions in the United States during year 2020. METHODS: The 2021 NBCUS survey instrument was modified to include certain blood collection and utilization variables for 2020. The survey was distributed to all US blood collection centers, all US hospitals performing ≥1000 surgeries annually, and a 40% random sample of hospitals performing 100-999 surgeries annually. Weighting and imputation were used to generate national estimates for whole blood and apheresis platelet donation; RBC and platelet transfusion; and convalescent plasma distribution. RESULTS: Whole blood collections were stable from 2019 (9,790,000 units; 95% CI: 9,320,000-10,261,000) to 2020 (9,738,000 units; 95% CI: 9,365,000-10,110,000). RBC transfusions decreased by 6.0%, from 10,852,000 units (95% CI: 10,444,000-11,259,000) in 2019 to 10,202,000 units (95% CI: 9,811,000-10,593,000) in 2020. Declines were steepest during March-April 2020, with transfusions subsequently rebounding. Apheresis platelet collections increased from 2,359,000 units (95% CI: 2,240,000-2,477,000) in 2019 to 2,408,000 units (95% CI: 2,288,000-2,528,000) in 2020. Apheresis platelet transfusions increased from 1,996,000 units (95% CI: 1,846,000-2,147,000) in 2019 to 2,057,000 units (95% CI: 1,902,000-2,211,000) in 2020. CONCLUSION: The COVID-19 pandemic resulted in reduced blood donations and transfusions in some months during 2020 but only a minimal annualized decline compared with 2019.

Proceedings of the 2022 NHLBI and OASH state of the science in transfusion medicine symposium.

BACKGROUND: State of the Science (SoS) meetings are used to define and highlight important unanswered scientific questions. The National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, and the Office of the Assistant Secretary for Health (OASH), Department of Health and Human Services held a virtual SoS in transfusion medicine (TM) symposium. STUDY DESIGN AND METHODS: In advance of the symposium, six multidisciplinary working groups (WG) convened to define research priorities in the areas of: blood donors and the supply, optimizing transfusion outcomes for recipients, emerging infections, mechanistic aspects of components and transfusion, new computational methods in transfusion science, and impact of health disparities on donors and recipients. The overall objective was to identify key basic, translational, and clinical research questions that will help to increase and diversify the volunteer donor pool, ensure safe and effective transfusion strategies for recipients, and identify which blood products from which donors best meet the clinical needs of specific recipient populations. RESULTS: On August 29-30, 2022, over 400 researchers, clinicians, industry experts, government officials, community members, and patient advocates discussed the research priorities presented by each WG. Dialogue focused on the five highest priority research areas identified by each WG and included the rationale, proposed methodological approaches, feasibility, and barriers for success. DISCUSSION: This report summarizes the key ideas and research priorities identified during the NHLBI/OASH SoS in TM symposium. The report highlights major gaps in our current knowledge and provides a road map for TM research.

IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings.

Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Šresolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition.

Continued stabilization of blood collections and transfusions in the United States: Findings from the 2021 National Blood Collection and Utilization Survey.

BACKGROUND: National Blood Collection and Utilization Surveys (NBCUS) have reported decreases in U.S. blood collections and transfusions since 2008. The declines began to stabilize in 2015-2017, with a subsequent increase in transfusions in 2019. Data from the 2021 NBCUS were analyzed to understand the current dynamics of blood collection and use in the United States. METHODS: In March 2022, all community-based (53) and hospital-based (83) blood collection centers, a randomly selected 40% of transfusing hospitals performing 100-999 annual inpatient surgeries, and all transfusing hospitals performing ≥1000 annual inpatient surgeries were sent a 2021 NBCUS survey to ascertain blood collection and transfusion data. Responses were compiled, and national estimates were calculated for the number of units of blood and blood components collected, distributed, transfused, and outdated in 2021. Weighting and imputation were applied to account for non-responses and missing data, respectively. RESULTS: Survey response rates were 92.5% (49/53) for community-based blood centers, 74.7% (62/83) for hospital-based blood centers, and 76.3% (2102/2754) for transfusing hospitals. Overall, 11,784,000 (95% confidence interval [CI], 11,392,000-12,177,000) whole blood and apheresis red blood cell (RBC) units were collected in 2021, a 1.7% increase from 2019; 10,764,000 (95% CI, 10,357,000-11,171,000) whole blood-derived and apheresis RBC units were transfused, a 0.8% decrease. Total platelet units distributed increased by 0.8%; platelet units transfused decreased by 3.0%; plasma units distributed increased by 16.2%; and plasma units transfused increased by 1.4%. DISCUSSION: The 2021 NBCUS findings demonstrate a stabilization in U.S. blood collections and transfusions, suggesting a plateau has been reached for both.

Dimensional Reduction for Single-Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67.

Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, methods that provide the resolution needed for complex kinetic studies of multicomponent condensation are desired. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the two-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from three-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a time scale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length, and GC content affect the threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1α and Ki-67, have much lower threshold concentrations for condensation than charge-based condensing agents, with Ki-67 being the most effective, requiring as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1α and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color are used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67-mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions.

Single-molecule dynamics of DNA gyrase in evolutionarily distant bacteria Mycobacterium tuberculosis and Escherichia coli.

DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection. Gyrase hydrolyzes ATP to add negative supercoils to DNA using a strand passage mechanism that has been investigated using biophysical and biochemical approaches. To analyze the dynamics of substeps leading to strand passage, single-molecule rotor bead tracking (RBT) has been used previously to follow real-time supercoiling and conformational transitions in Escherichia coli (EC) gyrase. However, RBT has not yet been applied to gyrase from other pathogenically relevant bacteria, and it is not known whether substeps are conserved across evolutionarily distant species. Here, we compare gyrase supercoiling dynamics between two evolutionarily distant bacterial species, MTB and EC. We used RBT to measure supercoiling rates, processivities, and the geometries and transition kinetics of conformational states of purified gyrase proteins in complex with DNA. Our results show that E. coli and MTB gyrases are both processive, with the MTB enzyme displaying velocities ∼5.5× slower than the EC enzyme. Compared with EC gyrase, MTB gyrase also more readily populates an intermediate state with DNA chirally wrapped around the enzyme, in both the presence and absence of ATP. Our substep measurements reveal common features in conformational states of EC and MTB gyrases interacting with DNA but also suggest differences in populations and transition rates that may reflect distinct cellular needs between these two species.

Etoposide promotes DNA loop trapping and barrier formation by topoisomerase II.

Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and cytotoxicity. How etoposide perturbs topoisomerase dynamics is not known. Here we investigated the action of etoposide on yeast topoisomerase II, human topoisomerase IIα and human topoisomerase IIß using several sensitive single-molecule detection methods. Unexpectedly, we found that etoposide induces topoisomerase to trap DNA loops, compacting DNA and restructuring DNA topology. Loop trapping occurs after ATP hydrolysis but before strand ejection from the enzyme. Although etoposide decreases the innate stability of topoisomerase dimers, it increases the ability of the enzyme to act as a stable roadblock. Interestingly, the three topoisomerases show similar etoposide-mediated resistance to dimer separation and sliding along DNA but different abilities to compact DNA and chirally relax DNA supercoils. These data provide unique mechanistic insights into the functional consequences of etoposide on topoisomerase II dynamics.

DNA-Stimulated Liquid-Liquid phase separation by eukaryotic topoisomerase ii modulates catalytic function.

Type II topoisomerases modulate chromosome supercoiling, condensation, and catenation by moving one double-stranded DNA segment through a transient break in a second duplex. How DNA strands are chosen and selectively passed to yield appropriate topological outcomes - for example, decatenation vs. catenation - is poorly understood. Here, we show that at physiological enzyme concentrations, eukaryotic type IIA topoisomerases (topo IIs) readily coalesce into condensed bodies. DNA stimulates condensation and fluidizes these assemblies to impart liquid-like behavior. Condensation induces both budding yeast and human topo IIs to switch from DNA unlinking to active DNA catenation, and depends on an unstructured C-terminal region, the loss of which leads to high levels of knotting and reduced catenation. Our findings establish that local protein concentration and phase separation can regulate how topo II creates or dissolves DNA links, behaviors that can account for the varied roles of the enzyme in supporting transcription, replication, and chromosome compaction.

Biochemical methods to monitor loading and activation of hexameric helicases.

Ring-shaped hexameric helicases are an essential class of enzymes that unwind duplex nucleic acids to support a variety of cellular processes. Because of their critical roles in cells, hexameric helicase dysfunction has been linked to DNA damage and genomic instability. Biochemical characterization of hexameric helicase activity and regulation in vitro is necessary for understanding enzyme function and aiding drug discovery efforts. In this chapter, we describe protocols for characterizing mechanisms of helicase loading, activation, and unwinding using the model replicative hexameric DnaB helicase and its cognate DnaC loading factor from E. coli.