Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA.

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.

Retroviral prototype foamy virus intasome binding to a nucleosome target does not determine integration efficiency.

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.

Preservice Observation in Special Education: A Validation Study.

Advancing teacher candidates' overall competence through use of valid teacher observation systems should be an essential element of teacher preparation. Yet, the field of special education has not provided observation protocols designed specifically for preservice teachers that are founded in theoretical perspectives and research on effective instruction for students with learning and other high-incidence disabilities (SWDs). To address this need, a group of researchers in special education teacher preparation and measurement developed the Preservice Observation Instrument for Special Education (POISE). The POISE is an observation system rooted in effective special education practices that support the growth of preservice teachers who will serve SWD across instructional settings. The purpose of this article is to report on the development and psychometric properties of the POISE. Specifically, we employed Kane's argument-based validity approach to frame each stage in the development process of the POISE. We conducted two phases of content validation activities, development activities, and a pilot study to assess the degree to which scores from POISE provided evidence for the scoring, generalizability, and extrapolation inferences. In the end, the POISE represents a promising observation instrument for the development of special education teacher candidates.

Danielson's Framework for Teaching: Convergence and Divergence With Conceptions of Effectiveness in Special Education.

Danielson's Framework for Teaching (FFT) is currently used in more than 20 states to inform teacher evaluation and professional learning. To investigate whether FFT promotes instruction that appropriately responds to the needs of students with learning disabilities, we conduct a systematic content analysis of the instructional approach emphasized in the FFT's Instructional Domain (Domain 3) of Danielson's FFT. We frame our study using cognitive load theory and research regarding effective instruction for students with disabilities. We end by discussing implications regarding the evaluation and development of effective teaching for students with learning disabilities.

Nucleosome DNA unwrapping does not affect prototype foamy virus integration efficiency or site selection.

Eukaryotic DNA binding proteins must access genomic DNA that is packaged into chromatin in vivo. During a productive infection, retroviral integrases (IN) must similarly interact with chromatin to integrate the viral cDNA genome. Here we examine the role of nucleosome DNA unwrapping in the retroviral integrase search for a target site. These studies utilized PFV intasomes that are comprised of a tetramer of PFV IN with two oligomers mimicking the viral cDNA ends. Modified recombinant human histones were used to generate nucleosomes with increased unwrapping rates at different DNA regions. These modifications included the acetylmimetic H3(K56Q) and the chemically engineered H4(K77ac, K79ac). While transcription factors and DNA damage sensors may search nucleosome bound DNA during transient unwrapping, PFV intasome mediated integration appears to be unaffected by increased nucleosome unwrapping. These studies suggest PFV intasomes do not utilize nucleosome unwrapping to search nucleosome targets.

Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid.

The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.

Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration.

Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion.

Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3'-5' direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3'-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an 'anchor' site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.

Synthesis, structure, electrochemistry, and electrochemiluminescence of thienyltriazoles.

Four blue-emitting thienyltriazoles with desired N and O coordination atoms were prepared in high yield via click chemistry for potential incorporation into metal complexes. Three of their crystal structures were determined by X-ray crystallography. The electrochemical properties, electronic structures of these thienyltriazoles, 1-4, and their correlations were studied using cyclic voltammetry and differential pulse voltammetry techniques along with density function theory (DFT) calculations. All of the compounds underwent irreversible redox reactions, leading to unstable electrogenerated radical cations and anions. Electrochemical gaps determined from the differences between first formal reduction and oxidation reactions were correlated to HOMO-LUMO energy gaps obtained from UV-vis spectroscopy and the DFT calculations as well as energies of excited states measured from photoluminescence spectroscopy. We observed weak electrochemiluminescence (ECL) from annihilation of these thienyltriazole radicals in acetonitrile containing 0.1 M tetra-n-butylammonium perchlorate as electrolyte. An enhancement in ECL efficiency ranging from 0.16 to 0.50% was observed upon addition of benzoyl peroxide as a coreactant in the above electrolyte solutions. The generation of excimers in solutions of 1-4 was observed as seen by the red-shift in ECL maxima relative to their corresponding photoluminescence peak wavelengths. Our work is of importance for the development of efficient blue-emitting fluorophores via click chemistry that could be potential luminophores in metal complexes.

An N-heterocyclic carbene containing a bithiophene backbone: synthesis and coordination chemistry.

A new N-heterocyclic carbene (NHC) containing a fused bithiophene backbone has been synthesized along with its silver(I) and BPh(3) complexes. The donor strength of this unique NHC has been determined from the IR stretching frequencies of the isolated NHC-Rh(CO)(2)Cl complex. The photophysical properties of all of the novel compounds have been investigated and are presented.

Chemistry of the heavy group 15 elements with the pyridyl tethered 1,2-bis(imino)acenaphthene "clamshell" ligand.

Cobaltocene has been used as a one-electron reductant in a facile route to generate pnictogen(I) (P, As) synthons. These subsequently undergo a formal 4 + 2 cycloaddition with a pyridyl tethered 1,2-bis(imino)acenaphthene "clamshell" ligand to yield N-heterocyclic chlorophosphines and -arsines, which are precursors to the corresponding N-heterocyclic pnictenium cations. In the absence of a reductant the "clamshell" ligand can be used in forming hypervalent donor-acceptor complexes with heavy main group elements (Sn, Sb and Bi).

Synthesis, reactivity, and computational analysis of halophosphines supported by dianionic guanidinate ligands.

The reported chemistry and reactivity of guanidinate supported group 15 elements in the +3 oxidation state, particularly phosphorus, is limited when compared to their ubiquity in supporting metallic elements across the periodic table. We have synthesized a series of chlorophosphines utilizing homo- and heteroleptic (dianionic)guanidinates and have completed a comprehensive study of their reactivity. Most notable is the reluctancy of these four-membered rings to form the corresponding N-heterocyclic phosphenium cations, the tendency to chemically and thermally eliminate carbodiimide, and the scarcely observed ring expansion by insertion of a chloro(imino)phosphine into a P-N bond of the P-N-C-N framework. Computational analysis has provided corroborating evidence for the unwillingness of the halide abstraction reaction by demonstrating the exceptional electron acceptor properties of the target phosphenium cations and the underscoring strength of the P-X bond.

Group 15 pnictenium cations supported by a conjugated bithiophene backbone.

Thiophene based polymers and oligomers have attracted considerable attention because they can be functionalized to alter the gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), which enables the design of tunable light emitting materials. One area, which has been less explored, is the incorporation of low coordinate, low oxidation state main group elements into these systems. We have currently developed a novel π-conjugated ligand containing two contiguous thiophene rings in which we have demonstrated its ability to support both pnictogen cations and their metal complexes.

Electrogenerated chemiluminescence of triazole-modified deoxycytidine analogues in N,N-dimethylformamide.

Triazole-modified deoxycytidines have been prepared for incorporation into single-stranded deoxyribonucleic acid (ssDNA). Electrochemical responses and electrogenerated chemiluminescence (ECL) of these deoxycytidine (dC) analogues, 1-4, were investigated as the monomers. Cyclic voltammetry and differential pulse voltammetry techniques were used to determine the oxidation and reduction potentials of 1-4, along with the reversibility of their electrochemical reactions. The dC analogues, in N,N-dimethylformamide containing 0.1 M tetra-n-butylammonium perchlorate as electrolyte, exhibited weak relative ECL efficiencies following the annihilation mechanism, while these efficiencies were enhanced with the use of benzoyl peroxide following the coreactant mechanism. It was shown that these nucleosides could generate excited monomers, and excimers as seen by the red-shifted ECL maxima relative to their corresponding photoluminescence peak wavelengths.

A new approach to internal Lewis pairs featuring a phosphenium acid and a pyridine base.

A bis(imino)acenaphthene (BIAN) ligand containing a pendant Lewis base has been used as a new framework to support a N-heterocyclic phosphenium cation (NHP). Reactivity studies demonstrate the ability of the ligand to act as a Lewis base, while the phosphorus centre provides a Lewis acidic site, giving new opportunities in NHP chemistry.

Blue fluorescent deoxycytidine analogues: convergent synthesis, solid-state and electronic structure, and solvatochromism.

We report the synthesis and photospectroscopic characterisation of intrinsically fluorescent triazole-appended cytidines. Fluorescence was found to be highly dependent on solvent conditions. X-Ray crystallographic data show the proton of the exocyclic amine of the nucleobase and the triazole N(3) engaged in a H-bond.

Hydrogelation abilities of nucleobase-modified cytidines possessing substituted triazoles.

Nucleoside-derived hydrogelators have been sought for their potential biomedical applications, such as are found in tissue engineering and drug delivery. By judiciously adding a degree of hydrophobicity certain analogues are able to form micelles, bi-layers and gels in water. Research in this area has yet to lay down solid ground rules for the rational design of novel nucleoside gelators making further studies necessary. The synthesis and examination of a series of aryl-substituted 5-triazolylcytidines yielded an analogue that gelates water. 5-(1-(2,2'-bithiophen-3-yl)-1H-1,2,3-triazol-4-yl)-2'-deoxycytidine was found to form gels in water down to 0.3 wt%. The ribocytidine analogue failed to form gel in aqueous solution; but was able to form a hydrogel in the presence of guanosine. Images obtained by SEM show the different architectures of the gel; varying from cribriform to fibrous to lamellar. The present gelating compound studied may have potential as a component of a controlled-release drug delivery system.

Synthesis of N,C bound sulfur, selenium, and tellurium heterocycles via the reaction of chalcogen halides with -CH3 substituted diazabutadiene ligands.

A series of N,C bound chalcogen heterocycles from the reaction of chalcogen halides (ChX(n); Ch = S, Se Te; X = Cl, Br; n = 2, 4) with N-alkyl or N-aryl 1,4-diazabutadiene (DAB) ligands featuring methyl substituents on the backbone C-C linkage are reported. In contrast to what is observed for other p-block elements with the same ligand systems, which typically bind in an N,N' fashion, the chalcogens react with the ligand in an unusual manner, forming N(1)C(3)Ch(1) five-membered rings by incorporating a "backbone" methyl group. Solid state structures of the feature compounds have been confirmed by X-ray crystallographic studies. The reaction mechanism was probed by deuterium isotope labeling of the DAB ligand and analyzed using stopped-flow kinetics experiments, which supported attack by the olefin in the enamine form of the DAB ligand with concomitant loss of HX.

Meta-substituted thienyl benzenes: a comparative synthetic, structural and computational study.

A selection of metal-catalyzed C-C bond-forming strategies has been evaluated in the synthesis of a series of meta-substituted thienylbenzenes, (T(n))(2)C(6)H(4) and (T(n))(3)C(6)H(3) (n = 1, 2; T(1) = 2-thienyl; T(2) = 2,2'-bithien-5-yl). Kumada coupling reactions catalyzed by PdCl(2)(dppf) between the appropriate thienyl Grignard and either 1,3- or 1,3,5-bromo- or iodobenzenes were found to be the most reliable in terms of convenience, selectivity and yield (dppf = 1,1'-bis(diphenylphosphino)ferrocene). These conditions also allowed the optimized syntheses of mixed (thienyl)(halo)benzenes, (T(n))C(6)H(3)X(2) and (T(n))(2)C(6)H(3)X (X = Br, I); the latter bromides could be further elaborated in subsequent Stille, Sonogashira, or Kumada reactions to furnish bis(thienyl) compounds bearing electron-donating or -withdrawing groups in the third meta position, (T(n))(2)C(6)H(3)R (R = Ph, p-MeOC(6)H(4), p-FC(6)H(4); n = 1, 2) and (T(1))(2)C(6)H(3)R' (R' = Me, C[triple bond]CPh, Fc; Fc = ferrocenyl). The relative effects of R, R', and n were evaluated by electronic spectroscopy, cyclic voltammetry and calculation. The absorption and emission characteristics and calculated ionization potentials and HOMO-LUMO gaps of these compounds were strongly dependent on n and largely insensitive to R/R'. These measured and calculated properties were also found to be largely invariant with respect to the degree of substitution about the central ring in the meta-substituted benzenes (T(n))(m)X(3-m)C(6)H(3) (m = 1-3; n = 1, 2; X = Br, H), although in the case of n = 1, there was a smooth, albeit small, increase in the emission maximum with increasing thienyl substitution. These findings essentially confirmed earlier theoretical predictions that the thienyl "arms" of meta-substituted phenyl-cored dendrimers were predominantly responsible for absorption and that excitons were localized to the "arms" without any electronic coupling between them, but also introduced the caveat that there was a minimum of two contiguous thiophene rings required for strict exciton localization to the arms. The oxidation potentials of the compounds in solution ranged from 0.9 to 1.6 V and were not rationally dependent on the degree of substitution or the nature of R/R'.