Janet E. Lindsley

Steady-state and Rapid Kinetic Analysis of Topoisomerase II Trapped as the Closed-clamp Intermediate by ICRF-193

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC50 of 6.5 ± 1 μm for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.

KINETIC AND THERMODYNAMIC ANALYSIS OF MUTANT TYPE II DNA TOPOISOMERASES THAT CANNOT COVALENTLY CLEAVE DNA

DNA topoisomerase II catalyzes two different chemical reactions as part of its DNA transport cycle: ATP hydrolysis and DNA breakage/religation. The coordination between these reactions was studied using mutants of yeast topoisomerase II that are unable to covalently cleave DNA. In the absence of DNA, the ATPase activities of these mutant enzymes are identical to the wild type activity. DNA binding stimulates the ATPase activity of the mutant enzymes, but with steady-state parameters different from those of the wild type enzyme. These differences were examined through DNA binding experiments and pre-steady-state ATPase assays. One mutant protein, Y782F, binds DNA with the same affinity as wild type protein. This mutant topologically traps one DNA circle in the presence of a nonhydrolyzable ATP analog under the same conditions that the wild type protein catenates two circles. Rapid chemical quench and pulse-chase ATPase experiments reveal that the mutant proteins bound to DNA have the same sequential hydrolysis reaction cycle as the wild type enzyme. Binding of ATP to the mutants is not notably impaired, but hydrolysis of the first ATP is slower than for the wild type enzyme. Models to explain these results in the context of the entire DNA topoisomerase II reaction cycle are discussed.

Yeast Topoisomerase II Is Inhibited by Etoposide After Hydrolyzing the First ATP and Before Releasing the Second ADP

Topoisomerase II-catalyzed DNA transport requires coordination between two distinct reactions: ATP hydrolysis and DNA cleavage/religation. To further understand how these reactions are coupled, inhibition by the clinically used anticancer drug etoposide was studied. The IC50 for perturbing the DNA cleavage/religation equilibrium is nucleotide-dependent; its value is 6 μm in the presence of ATP, 25 μm in the presence of a nonhydrolyzable ATP analog, and 45 μm in the presence of ADP or no nucleotide. This inhibition was further characterized using steady-state and pre-steady-state ATPase and decatenation assays. Etoposide is a hyperbolic noncompetitive inhibitor of the ATPase activity with aK i (app) of 5.6 μm; no inhibition of ATP hydrolysis is seen in the absence of DNA cleavage. In order to determine which steps of the ATPase mechanism etoposide inhibits, pre-steady-state analysis was performed. These results showed that etoposide does not reduce the rate of binding two ATP, hydrolyzing the first ATP, or releasing the second ADP. Inhibition is therefore associated with the first product release step or hydrolysis of the second ATP, suggesting that DNA religation normally occurs at one of these two steps. Multiple turnover decatenation is inhibited when etoposide is present; however, single turnover decatenation occurs normally. The implications of these results are discussed in terms of their contribution to our current model for the topoisomerase II mechanism.

Revealing the allosterome: systematic identification of metabolite-protein interactions.

Small molecule allostery modifies protein function but is not easily discovered. We introduce mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS), a method for identifying physiologically relevant, low-affinity metabolite-protein interactions using unmodified proteins and complex mixtures of unmodified metabolites. In a pilot experiment using five proteins, we identified 16 known and 13 novel interactions. The known interactions included substrates, products, intermediates, and allosteric regulators of their protein partners. MIDAS does not depend upon enzymatic measurements, but most of the new interactions affect the enzymatic activity of the protein partner. We found that the fatty acid palmitate interacts with both glucokinase and glycogen phosphorylase. Further characterization revealed that palmitate inhibited both enzymes, possibly providing a mechanism for sparing carbohydrate catabolism when fatty acids are abundant.

The Two-Stage Examination: A Method to Assess Individual Competence and Collaborative Problem Solving in Medical Students.

Problem Effectively solving problems as a team under stressful conditions is central to medical practice; however, because summative examinations in medical education must test individual competence, they are typically solitary assessments. Approach Using two-stage examinations, in which students first answer questions individually (Stage 1) and then discuss them in teams prior to resubmitting their answers (Stage 2), is one method for rectifying this discordance. On the basis of principles of social constructivism, the authors hypothesized that two-stage examinations would lead to better retention of, specifically, items answered incorrectly at Stage 1. In fall 2014, they divided 104 first-year medical students into two groups of 52 students. Groups alternated each week between taking one- and two-stage examinations such that each student completed 6 one-stage and 6 two-stage examinations. The authors reassessed 61 concepts on a final examination and, using the Wilcoxon signed ranked tests, compared performance for all concepts and for just those students initially missed, between Stages 1 and 2. Outcomes Final examination performance on all previously assessed concepts was not significantly different between the one-and two-stage conditions (P = .77); however, performance on only concepts that students initially answered incorrectly on a prior examination improved by 12% for the two-stage condition relative to the one-stage condition (P = .02, r = 0.17). Next Steps Team assessment may be most useful for assessing concepts students find difficult, as opposed to all content. More research is needed to determine whether these results apply to all medical school topics and student cohorts.

What's in a Transition? An Integrative Perspective on Transitions in Medical Education

ABSTRACT This Conversation Starters article presents a selected research abstract from the 2016 Association of American Medical Colleges Western Region Group on Educational Affairs annual spring meeting. The abstract is paired with the integrative commentary of three experts who shared their thoughts stimulated by the needs assessment study. These thoughts explore how the general theoretical mechanisms of transition may be integrated with cognitive load theory in order to design interventions and environments that foster transition.

Type II DNA topoisomerases: Coupling directional DNA transport to ATP hydrolysis

Publisher Summary This chapter describes the mechanism of the type II enzymes and focuses on the present knowledge about the way these enzymes couple adenosine triphosphate (ATP) binding and hydrolysis to the directional transport of one duplex DNA segment through a transient break in another. Type II topoisomerases are essential in all organisms for unlinking replicated chromosomes and establishing the proper condensed state of these chromosomes. These enzymes are also the targets of many natural and man-made toxins, some of which are used as antimicrobials and chemotherapeutics. Type II topoisomerases require ATP to catalyze the directional transport of a T segment through a G segment. This directionality has been long appreciated for gyrase, the prokaryotic type II topoisomerase that uniquely introduces negative supercoils into DNA. Because gyrase increases the free energy of its DNA substrate, it is obvious why it requires ATP. However, the other type II topoisomerases, including all the known eukaryotic type IIA proteins, the bacteriophage enzymes, and prokaryotic topo IV cannot supercoil DNA. These enzymes, referred to as “topo II/IV” to distinguish them from gyrase, can all relax supercoiled DNA, bringing it to a lower-energy state, but cannot supercoil it. They can also catenate/decatenate and knot/unknot DNA circles.

Does It Take Two to Untangle

The structural integrity of mitotic chromosomes is essential for proper chromatid segregation. In this issue of Cell, show that vertebrates contain two distinct condensin complexes, both of which are required for normal mitotic chromosome morphology.