Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry.

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl ß,γ-imidodiphosphate or the anticancer drug ICRF-193.

The interface of transcription and DNA replication in the mitochondria.

DNA replication of the mitochondrial genome is unique in that replication is not primed by RNA derived from dedicated primases, but instead by extension of processed RNA transcripts laid down by the mitochondrial RNA polymerase. Thus, the RNA polymerase serves not only to generate the transcripts but also the primers needed for mitochondrial DNA replication. The interface between this transcription and DNA replication is not well understood but must be highly regulated and coordinated to carry out both mitochondrial DNA replication and transcription. This review focuses on the extension of RNA primers for DNA replication by the replication machinery and summarizes the current models of DNA replication in mitochondria as well as the proteins involved in mitochondrial DNA replication, namely, the DNA polymerase γ and its accessory subunit, the mitochondrial DNA helicase, the single-stranded DNA binding protein, topoisomerase I and IIIα and RNaseH1. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective.

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate.

Creating and sustaining collaborative multi-institutional industry site visit programs: a toolkit.

Background: As more early career scientists enter into diverse career pathways, visiting local companies or organizations can support their exploration of these paths. As an efficient way to facilitate this, we developed a collaborative regional site visit program: the Enhancing Local Industry Transitions through Exploration (ELITE) Consortium.  Consortium members arrange half-day visits to local industry sites, thus providing companies and trainees the opportunity to meet and identify potential professional and career opportunities. Three different training institutions worked cooperatively in the development and maintenance of the program. The ELITE Consortium was developed with eight phased steps; guidelines and operating procedures were created for each of these steps and are provided along with sample materials for institutions interested in building similar programs. Methods: Prior to fully developing the program, trainee interests were evaluated via questionnaire. During program implementation and thereafter, program directors tracked attendance and collected career outcome data from publicly available sources to identify first job positions after training. Regression analyses and chi-squared analyses were used to examine site visit matches and career outcome data. Results: Analyses suggest a positive impact of site visits on postdoctoral and graduate trainees' career outcomes at companies or institutions that match a similar sector (e.g., for-profit) and type (e.g., biotech, pharmaceutical, contract research organization). Despite a small sample size, evidence suggests an especially positive impact on trainees who organize site visits to companies compared with those who simply participate. Conclusions: The ELITE Consortium was successful in helping trainees explore and identify a multitude of career paths. Trainees attained employment either directly or in related companies and institutions visited by ELITE participants. The joint, three-institution, flexible nature of the ELITE Consortium positively impacts the program's sustainability and reach. The toolkit provided here will help other institutions to replicate and adapt the program with minimal effort.

Monitoring the topoisomerase II DNA gate conformational change with fluorescence resonance energy transfer.

Type II DNA topoisomerases are essential, ubiquitous enzymes responsible for performing vital functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) performs these DNA transactions by passing one segment of DNA through the other using a reversible, enzyme-bridged double-stranded DNA break. This cleavage/religation of the DNA backbone is coupled to the opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle. To monitor the opening and closing of the DNA gate, we designed an oligonucleotide substrate with a pair of fluorophores flanking the topoisomerase II cleavage site, such that the fluorophores undergo efficient fluorescence resonance energy transfer (FRET) in the intact DNA substrate, but the FRET efficiency decreases as topo II opens the DNA gate. Here we present a method for creating the DNA substrate and using it as a tool to monitor the conformational changes at the topo II DNA gate. We detail how to collect and process fluorescence spectra to determine the FRET efficiency of the DNA substrate.

Single-molecule measurements of the opening and closing of the DNA gate by eukaryotic topoisomerase II.

Type II DNA topoisomerases are essential and ubiquitous enzymes that perform important functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerases carry out these DNA transactions by passing one segment of DNA through the other by using a reversible, enzyme-bridged double strand break. The transient enzyme/DNA adduct is mediated by a phosphodiester bond between the active-site tyrosine and a backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this cleavage/religation. We designed a unique oligonucleotide substrate with a pair of fluorophores straddling the topoisomerase II cleavage site, allowing the use of FRET to monitor the opening of the DNA gate. The DNA substrate undergoes an enzyme-mediated transition between a closed and open state in the presence of ATP, similar to the overall topoisomerase II catalyzed reaction. Single-molecule fluorescence microscopy measurements demonstrate that the transition has comparable rate constants for both the opening and closing reaction during steady-state ATP hydrolysis, with an apparent equilibrium constant near unity. In the presence of AMPPNP, a reduction in FRET occurs, suggesting an opening or partial opening of the DNA gate. However, the single-molecule experiments indicate that the open and closed states do not interconvert at a measurable rate.