Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

KERA: analysis tool for multi-process, multi-state single-molecule data.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.

Dynamic elements of replication protein A at the crossroads of DNA replication, recombination, and repair.

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.

Analysis of regional scale risk of whirling disease in populations of Colorado and Rio Grande cutthroat trout using a Bayesian belief network model.

Introduction and spread of the parasite Myxobolus cerebralis, the causative agent of whirling disease, has contributed to the collapse of wild trout populations throughout the intermountain west. Of concern is the risk the disease may have on conservation and recovery of native cutthroat trout. We employed a Bayesian belief network to assess probability of whirling disease in Colorado River and Rio Grande cutthroat trout (Oncorhynchus clarkii pleuriticus and Oncorhynchus clarkii virginalis, respectively) within their current ranges in the southwest United States. Available habitat (as defined by gradient and elevation) for intermediate oligochaete worm host, Tubifex tubifex, exerted the greatest influence on the likelihood of infection, yet prevalence of stream barriers also affected the risk outcome. Management areas that had the highest likelihood of infected Colorado River cutthroat trout were in the eastern portion of their range, although the probability of infection was highest for populations in the southern, San Juan subbasin. Rio Grande cutthroat trout had a relatively low likelihood of infection, with populations in the southernmost Pecos management area predicted to be at greatest risk. The Bayesian risk assessment model predicted the likelihood of whirling disease infection from its principal transmission vector, fish movement, and suggested that barriers may be effective in reducing risk of exposure to native trout populations. Data gaps, especially with regard to location of spawning, highlighted the importance in developing monitoring plans that support future risk assessments and adaptive management for subspecies of cutthroat trout.

An Atomistic Understanding of Allosteric Inhibition of Glutamate Racemase: a Dampening of Native Activation Dynamics.

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.

Dynamics and selective remodeling of the DNA-binding domains of RPA.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA-interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.

Assessment of exposure risk of polychlorinated biphenyls to interior least terns (Sterna antillarum).

Risk of polychlorinated biphenyl (PCB) exposure and effects were assessed for a colony of federally endangered interior least terns (Sterna antillarum) nesting on the Bitter Lake National Wildlife Refuge (NM, USA). The colony feeds from an area on the Refuge (Hunter Marsh/Oxbow Complex) wherein fish with elevated concentrations of total PCBs have been documented. Concentrations of total PCBs in whole fish averaged 0.94 mg/kg with a maximum concentration of 2.77 mg/kg, wet weight. Estimated daily PCB intake rates by adult birds throughout their 180-d breeding season ranged from <0.01 mg/kg/d to 0.98 mg/ kg/d, yielding hazard quotients that ranged from 0.01 to 21.68. Polychlorinated biphenyls pose a moderate risk to the colony of interior least terns that breed at the Bitter Lake National Wildlife Refuge, but the exposure rate is not likely to threaten their overall breeding success.

A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA.

Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Šresolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.

Effects of depleted uranium on survival, growth, and metamorphosis in the African clawed frog (Xenopus laevis).

Embryos (stage 8-47, Nieuwkoop and Faber) of the African clawed frog (Xenopus laevis) were subjected to water-borne depleted uranium (DU) concentrations that ranged from 4.8 to 77.7 mg/L using an acute 96-h frog embryo teratogenesis assay-Xenopus (FETAX). In a chronic 64-d assay, X. laevis (from embryo through metamorphosis; stages 8-66) were subjected to concentrations of DU that ranged from 6.2 to 54.3 mg/L. Our results indicate DU is a non teratogenic metal. No effects on mortality, malformations, or growth were observed in the 96-h FETAX with concentrations of DU that ranged from 4.8 to 77.7 mg/L. From stage 8 to stage 47, X. laevis tadpoles do not actively feed and the gills are not well developed. Thus, uptake of DU was reduced despite exposure to elevated concentrations. The 64-d assay resulted in no concentration response for either mortality or malformations; however, a delay in metamorphosis was observed in tadpoles subjected to elevated DU concentrations (from 13.1 to 54.3 mg/L) compared to tadpoles in both the well-water control and reference. The delay in metamorphosis was likely due to increasing body burden of DU that ranged from 0.98 to 2.82 mg/kg.

Effects of depleted uranium on the health and survival of Ceriodaphnia dubia and Hyalella azteca.

Depleted uranium (DU) has been used as a substitute for the fissionable enriched uranium component of atomic weapons tested at Los Alamos National Laboratory (LANL) (Los Alamos, NM, USA) since the early 1950s, resulting in considerable concentrations of DU in the soils within the test sites. Although the movement of DU into major aquatic systems has been shown to be minimal, there are many small-order ephemeral streams and areas of standing water in canyons throughout LANL that may be affected by inputs of DU via runoff, erosion, and leaching. Ninety-six-hour acute and 7-d chronic toxicity assays were conducted to measure the toxicity of DU on survival and reproduction of Ceriodaphnia dubia. A 14-d water-only assay was conducted to measure survival and growth of Hyalella azteca. The estimated median lethal concentration (LC50) to produce 50% mortality of the test population for the 96-h Ceriodaphnia dubia assay was 10.50 mg/L. Reproductive effects occurred at a lowest-observable-effect concentration > or = 3.91 mg/L with a no-observable-effect concentration of 1.97 mg/L. The estimated 14-d LC50 for the Hyalella azteca assay was 1.52 mg/L. No significant relationship was detected between growth and DU concentrations. Concentrations at which toxicity effects were observed in this study for both invertebrates exceeded concentrations of total uranium observed in runoff from LANL lands. Thus, it is likely that current runoff levels of uranium do not pose a threat to these types of aquatic invertebrates.

Concentration and dry deposition of mercury species in arid south central New Mexico (2001-2002).

This research was initiated to characterize atmospheric deposition of reactive gaseous mercury (RGM), particulate mercury (HgP; <2.5 microm), and gaseous elemental mercury (Hg0) in the arid lands of south central New Mexico. Two methods were field-tested to estimate dry deposition of three mercury species. A manual speciation sampling train consisting of a KCl-coated denuder, 2.5 microm quartz fiber filters, and gold-coated quartz traps and an ion-exchange membrane (as a passive surrogate surface) were deployed concurrently over 24-h intervals for an entire year. The mean 24-h atmospheric concentration for RGM was 6.8 pg m(-3) with an estimated deposition of 0.10 ng m(-2) h(-1). The estimated deposition of mercury to the passive surrogate surface was much greater (4.0 ng m(-2) h(-1)) but demonstrated a diurnal pattern with elevated deposition from late afternoon to late evening (1400-2200; 8.0 ng m(-2) h(-1)) and lowest deposition during the night just prior to sunrise (2200-0600; 1.7 ng m(-2) h(-1)). The mean 24-h atmospheric concentrations for HgP and Hg0 were 1.52 pg m(-3) and 1.59 ng m(-3), respectively. Diurnal patterns were observed for RGM with atmospheric levels lowest during the night prior to sunrise (3.8 pg m(-3)) and greater during the afternoon and early evening (8.9 pg m(-3)). Discernible diurnal patterns were not observed for either HgP or Hg0. The total dry deposition of Hg was 5.9 microg m-2 year-' with the contribution from the three species as follows: RGM (0.88 microg m(-2) year(-1)), HgP (0.025 microg m(-2) year(-1)), and Hg0 (5.0 microg m(-2) year(-1)). The annual wet deposition for total mercury throughout the same collection duration was 4.2 microg m(-2) year (-1), resulting in an estimated total deposition of 10.1 microg m(-2) year(-1) for Hg. On one sampling date, enhanced HgP (12 pg m(-3)) was observed due to emissions from a wildfire approximately 250 km to the east.

Inhibition of erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity in fish from waters affected by lead smelters.

We assessed the effects on fish of lead (Pb) released to streams by smelters located in Trail, BC (Canada), E. Helena, MT, Herculaneum, MO, and Glover, MO. Fish were collected by electrofishing from sites located downstream of smelters and from reference sites. Blood from each fish was analyzed for delta-aminolevulinic acid dehydratase (ALAD) activity and hemoglobin (Hb), and samples of blood, liver, or carcass were analyzed for Pb, zinc (Zn), or both. Fish collected downstream of all four smelters sites had elevated Pb concentrations, decreased ALAD activity, or both relative to their respective reference sites. At E. Helena, fish from the downstream site also had lower Hb concentrations than fish from upstream. Differences among taxa were also apparent. Consistent with previous studies, ALAD activity in catostomids (Pisces: Catostomidae-northern hog sucker, Hypentelium nigricans; river carpsucker, Carpiodes carpio; largescale sucker, Catostomus macrocheilus; and mountain sucker, C. platyrhynchus) seemed more sensitive to Pb-induced ALAD inhibition than the salmonids (Pisces: Salmonidae-rainbow trout, Oncorhynchus mykiss; brook trout, Salvelinus fontinalis) or common carp (Cyprinus carpio). Some of these differences may have resulted from differential accumulation of Zn, which was not measured at all sites. We detected no ALAD activity in channel catfish (Ictalurus punctatus) from either site on the Mississippi River at Herculaneum. MO. Our findings confirmed that Pb is released to aquatic ecosystems by smelters and accumulated by fish, and we documented potentially adverse effects of Pb in fish. We recommend that Zn be measured along with Pb when ALAD activity is used as a biomarker and the collection of at least 10 fish of a species at each site to facilitate statistical analysis.