Rapid tolerance to behavioral effects of ethanol in rats: Prevention by R-(-)-ketamine.

R-(-)-ketamine has emerged as a potentially improved medication over that of the (S)-isomer (marketed as Spravato for depression). Recent data have suggested (R)-ketamine could have value in the treatment of substance use disorder. The present set of experiments was undertaken to examine whether (R)-ketamine might prevent tolerance development. Rapid ethanol (ETOH) tolerance was studied since racemic ketamine had previously been shown to block this tolerance development in rats. In the present study, male Sprague-Dawley rats were given two large doses of ETOH on Day 1 (2.3 + 1.7 g/kg) and 2.3 g/kg ETOH on Day 2. Animals were tested for effects of 2.3 g/kg ETOH on grip strength, inclined screen performance and rotarod performance on Day 1 with or without (R)-ketamine as a pretreatment. (R)-ketamine alone was tested at the highest dose studied (10 mg/kg) and did not significantly influence any dependent measure. (R)-ketamine (1-10 mg/kg) did not alter the acute effects of ETOH except for enhancing the effects of ETOH on the inclined screen test at 3 mg/kg. Between-subjects analysis documented that tolerance developed to the effects of ETOH only on the measure of grip strength. (R)-ketamine (3 mg/kg) given prior to ETOH on Day 1 exhibited a strong trend toward preventing tolerance development (p = 0.062). The present results extend prior findings on the potential value of (R)-ketamine in substance abuse disorder therapeutics and add to the literature on NMDA receptor blockade as a tolerance-regulating mechanism.

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase.

Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

Regulation of Hed1 and Rad54 binding during maturation of the meiosis-specific presynaptic complex.

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.

Single-Stranded DNA Curtains for Studying the Srs2 Helicase Using Total Internal Reflection Fluorescence Microscopy.

Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments.

During meiosis, the two DNA recombinases Rad51 and Dmc1 form specialized presynaptic filaments that are adapted for performing recombination between homologous chromosomes. There is currently a limited understanding of how these two recombinases are organized within the meiotic presynaptic filament. Here, we used single molecule imaging to examine the properties of presynaptic complexes composed of both Rad51 and Dmc1. We demonstrate that Rad51 and Dmc1 have an intrinsic ability to self-segregate, even in the absence of any other recombination accessory proteins. Moreover, we found that the presence of Dmc1 stabilizes the adjacent Rad51 filaments, suggesting that cross-talk between these two recombinases may affect their biochemical properties. Based upon these findings, we describe a model for the organization of Rad51 and Dmc1 within the meiotic presynaptic complex, which is also consistent with in vivo observations, genetic findings, and biochemical expectations. This model argues against the existence of extensively intermixed filaments, and we propose that Rad51 and Dmc1 have intrinsic capacities to form spatially distinct filaments, suggesting that additional recombination cofactors are not required to segregate the Rad51 and Dmc1 filaments.

A change of view: homologous recombination at single-molecule resolution.

Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.

Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2.

Srs2 is a superfamily 1 (SF1) helicase and antirecombinase that is required for genome integrity. However, the mechanisms that regulate Srs2 remain poorly understood. Here, we visualize Srs2 as it acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. We demonstrate that Srs2 is a processive translocase capable of stripping thousands of Rad51 molecules from ssDNA at a rate of ∼50 monomers/s. We show that Srs2 is recruited to RPA clusters embedded between Rad51 filaments and that multimeric arrays of Srs2 assemble during translocation on ssDNA through a mechanism involving iterative Srs2 loading events at sites cleared of Rad51. We also demonstrate that Srs2 acts on heteroduplex DNA joints through two alternative pathways, both of which result in rapid disruption of the heteroduplex intermediate. On the basis of these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids.

An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance.

Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene.

Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of l0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.