All tangled up: how cells direct, manage and exploit topoisomerase function.

Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.

Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers.

Boron is essential for plant growth because of its incorporation into plant cell walls; however, in excess it is toxic to plants. Boron transport and homeostasis in plants is regulated in part by the borate efflux transporter Bor1, a member of the solute carrier (SLC) 4 transporter family with homology to the human bicarbonate transporter Band 3. Here, we present the 4.1-Å resolution crystal structure of Arabidopsis thaliana Bor1. The structure displays a dimeric architecture in which dimerization is mediated by centralized Gate domains. Comparisons with a structure of Band 3 in an outward-open state reveal that the Core domains of Bor1 have rotated inwards to achieve an occluded state. Further structural comparisons with UapA, a xanthine transporter from the nucleobase-ascorbate transporter family, show that the downward pivoting of the Core domains relative to the Gate domains may access an inward-open state. These results suggest that the SLC4, SLC26, and nucleobase-ascorbate transporter families all share an elevator transport mechanism in which alternating access is provided by Core domains that carry substrates across a membrane.

Thermodynamic system drift in protein evolution.

Proteins from thermophiles are generally more thermostable than their mesophilic homologs, but little is known about the evolutionary process driving these differences. Here we attempt to understand how the diverse thermostabilities of bacterial ribonuclease H1 (RNH) proteins evolved. RNH proteins from Thermus thermophilus (ttRNH) and Escherichia coli (ecRNH) share similar structures but differ in melting temperature (T(m)) by 20 °C. ttRNH's greater stability is caused in part by the presence of residual structure in the unfolded state, which results in a low heat capacity of unfolding (ΔCp) relative to ecRNH. We first characterized RNH proteins from a variety of extant bacteria and found that Tm correlates with the species' growth temperatures, consistent with environmental selection for stability. We then used ancestral sequence reconstruction to statistically infer evolutionary intermediates along lineages leading to ecRNH and ttRNH from their common ancestor, which existed approximately 3 billion years ago. Finally, we synthesized and experimentally characterized these intermediates. The shared ancestor has a melting temperature between those of ttRNH and ecRNH; the T(m)s of intermediate ancestors along the ttRNH lineage increased gradually over time, while the ecRNH lineage exhibited an abrupt drop in Tm followed by relatively little change. To determine whether the underlying mechanisms for thermostability correlate with the changes in T(m), we measured the thermodynamic basis for stabilization--ΔCp and other thermodynamic parameters--for each of the ancestors. We observed that, while the T(m) changes smoothly, the mechanistic basis for stability fluctuates over evolutionary time. Thus, even while overall stability appears to be strongly driven by selection, the proteins explored a wide variety of mechanisms of stabilization, a phenomenon we call "thermodynamic system drift." This suggests that even on lineages with strong selection to increase stability, proteins have wide latitude to explore sequence space, generating biophysical diversity and potentially opening new evolutionary pathways.

A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases.

Type II topoisomerases are required for the management of DNA tangles and supercoils, and are targets of clinical antibiotics and anti-cancer agents. These enzymes catalyse the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or 'gates'. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyse G-segment cleavage. Recent efforts have proposed that strand scission relies on a 'two-metal mechanism', a ubiquitous biochemical strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5A resolution, revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking experiments indicate that cleavage is catalysed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a molecular 'trapdoor' to prevent subunit dissociation during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.

OLD family nuclease function across diverse anti-phage defense systems.

Bacteriophages constitute a ubiquitous threat to bacteria, and bacteria have evolved numerous anti-phage defense systems to protect themselves. These systems include well-studied phenomena such as restriction endonucleases and CRISPR, while emerging studies have identified many new anti-phage defense systems whose mechanisms are unknown or poorly understood. Some of these systems involve overcoming lysogenization defect (OLD) nucleases, a family of proteins comprising an ABC ATPase domain linked to a Toprim nuclease domain. Despite being discovered over 50 years ago, OLD nuclease function remained mysterious until recent biochemical, structural, and bioinformatic studies revealed that OLD nucleases protect bacteria by functioning in diverse anti-phage defense systems including the Gabija system and retrons. In this review we will highlight recent discoveries in OLD protein function and their involvement in multiple discrete anti-phage defense systems.

Borate Transporters and SLC4 Bicarbonate Transporters Share Key Functional Properties.

Borate transporters are membrane transport proteins that regulate intracellular borate levels. In plants, borate is a micronutrient essential for growth but is toxic in excess, while in yeast, borate is unnecessary for growth and borate export confers tolerance. Borate transporters share structural homology with human bicarbonate transporters in the SLC4 family despite low sequence identity and differences in transported solutes. Here, we characterize the S. cerevisiae borate transporter Bor1p and examine whether key biochemical features of SLC4 transporters extend to borate transporters. We show that borate transporters and SLC4 transporters share multiple properties, including lipid-promoted dimerization, sensitivity to stilbene disulfonate-derived inhibitors, and a requirement for an acidic residue at the solute binding site. We also identify several amino acids critical for Bor1p function and show that disease-causing mutations in human SLC4A1 will eliminate in vivo function when their homologous mutations are introduced in Bor1p. Our data help elucidate mechanistic features of Bor1p and reveal significant functional properties shared between borate transporters and SLC4 transporters.

Human SLC4A11 does not complement BOR1 or support borate transport in Saccharomyces cerevisiae.

Borate is an essential micronutrient in plants regulated by borate transporters, which also protect both yeast and plants from toxically high levels of borate and share homology with the human SLC4 transporters. SLC4A11 is linked to congenital hereditary endothelial dystrophy and was initially reported to transport borate before subsequent studies rebutted this conclusion. To better understand the transport activities of purported borate transporters, we tested the ability of SLC4A11 and eleven borate transporters from A. thaliana and O. sativa to complement a BOR1 deletion in S. cerevisiae . We show that AtBOR4 , AtBOR5 , AtBOR7 , OsBOR2 , and OsBOR3 can each complement ScBOR1 , while the rest of the transporters tested do not rescue growth. Additionally, quantification of intracellular borate content demonstrates that SLC4A11 does not export borate in yeast, supporting studies that its transported substrate is not borate.

A biochemical approach to identifying microRNA targets.

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway.

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity.

Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.

The structure of DNA-bound human topoisomerase II alpha: conformational mechanisms for coordinating inter-subunit interactions with DNA cleavage.

Type II topoisomerases are required for the management of DNA superhelicity and chromosome segregation, and serve as frontline targets for a variety of small-molecule therapeutics. To better understand how these enzymes act in both contexts, we determined the 2.9-Å-resolution structure of the DNA cleavage core of human topoisomerase IIα (TOP2A) bound to a doubly nicked, 30-bp duplex oligonucleotide. In accord with prior biochemical and structural studies, TOP2A significantly bends its DNA substrate using a bipartite, nucleolytic center formed at an N-terminal dimerization interface of the cleavage core. However, the protein also adopts a global conformation in which the second of its two inter-protomer contact points, one at the C-terminus, has separated. This finding, together with comparative structural analyses, reveals that the principal site of DNA engagement undergoes highly quantized conformational transitions between distinct binding, cleavage, and drug-inhibited states that correlate with the control of subunit-subunit interactions. Additional consideration of our TOP2A model in light of an etoposide-inhibited complex of human topoisomerase IIß (TOP2B) suggests possible modification points for developing paralog-specific inhibitors to overcome the tendency of topoisomerase II-targeting chemotherapeutics to generate secondary malignancies.