Analysis of epigenetic features characteristic of L1 loci expressed in human cells.

Only a select few L1 loci in the human genome are expressed in any given cell line or organ, likely to minimize damage done to the genome. The epigenetic features and requirements of expressed L1 loci are currently unknown. Using human cells and comprehensive epigenetic analysis of individual expressed and unexpressed L1 loci, we determined that endogenous L1 transcription depends on a combination of epigenetic factors, including open chromatin, activating histone modifications, and hypomethylation at the L1 promoter. We demonstrate that the L1 promoter seems to require interaction with enhancer elements for optimal function. We utilize epigenetic context to predict the expression status of L1Hs loci that are poorly mappable with RNA-Seq. Our analysis identified a population of 'transitional' L1 loci that likely have greater potential to be activated during the epigenetic dysregulation seen in tumors and during aging because they are the most responsive to targeted CRISPR-mediated delivery of trans-activating domains. We demonstrate that an engineered increase in endogenous L1 mRNA expression increases Alu mobilization. Overall, our findings present the first global and comprehensive analysis of epigenetic status of individual L1 loci based on their expression status and demonstrate the importance of epigenetic context for L1 expression heterogeneity.

Altered DNA repair creates novel Alu/Alu repeat-mediated deletions.

Alu elements are the most abundant source of nonallelic homology that influences genetic instability in the human genome. When there is a DNA double-stranded break, the Alu element's high copy number, moderate length and distance and mismatch between elements uniquely influence recombination processes. We utilize a reporter-gene assay to show the complex influence of Alu mismatches on Alu-related repeat-mediated deletions (RMDs). The Alu/Alu heteroduplex intermediate can result in a nonallelic homologous recombination (HR). Alternatively, the heteroduplex can result in various DNA breaks around the Alu elements caused by competing nucleases. These breaks can undergo Alt-nonhomologous end joining to cause deletions focused around the Alu elements. Formation of these heteroduplex intermediates is largely RAD52 dependent. Cells with low ERCC1 levels utilize more of these alternatives resolutions, while cells with MSH2 defects tend to have more RMDs with a specific increase in the HR events. Therefore, Alu elements are expected to create different forms of deletions in various cancers depending on a number of these DNA repair defects.

A comprehensive approach to expression of L1 loci.

L1 elements represent the only currently active, autonomous retrotransposon in the human genome, and they make major contributions to human genetic instability. The vast majority of the 500 000 L1 elements in the genome are defective, and only a relatively few can contribute to the retrotransposition process. However, there is currently no comprehensive approach to identify the specific loci that are actively transcribed separate from the excess of L1-related sequences that are co-transcribed within genes. We have developed RNA-Seq procedures, as well as a 1200 bp 5΄ RACE product coupled with PACBio sequencing that can identify the specific L1 loci that contribute most of the L1-related RNA reads. At least 99% of L1-related sequences found in RNA do not arise from the L1 promoter, instead representing pieces of L1 incorporated in other cellular RNAs. In any given cell type a relatively few active L1 loci contribute to the 'authentic' L1 transcripts that arise from the L1 promoter, with significantly different loci seen expressed in different tissues.

New Ligand Design Provides Delocalization and Promotes Strong Absorption throughout the Visible Region in a Ru(II) Complex.

The new Ru(II)-anthraquinone complex [Ru(bpy)2(qdpq)](PF6)2 (Ru-qdpq; bpy = 2,2'-bipyridine; qdpq = 2,3-di(2-pyridyl)naphtho[2,3-f]quinoxaline-7,12-quinone) possesses a strong 1MLCT Ru → qdpq absorption with a maximum at 546 nm that tails into the near-IR and is significantly red-shifted relative to that of the related complex [Ru(bpy)2(qdppz)](PF6)2 (Ru-qdppz; qdppz = naphtho[2,3-a]dipyrido[3,2-h:2',3'-f]phenazine-5,18-dione), with λmax = 450 nm. Ru-qdppz possesses electronically isolated proximal and distal qdppz-based excited states; the former is initially generated and decays to the latter, which repopulates the ground state with τ = 362 ps. In contrast, excitation of Ru-qdpq results in the population of a relatively long-lived (τ = 19 ns) Ru(dπ) → qdpq(π*) 3MLCT excited state where the promoted electron is delocalized throughout the qdpq ligand. Ultrafast spectroscopy, used together with steady-state absorption, electrochemistry, and DFT calculations, indicates that the unique coordination modes of the qdpq and qdppz ligands impart substantially different electronic communication throughout the quinone-containing ligand, affecting the excited state and electron transfer properties of these molecules. These observations create a pathway to synthesize complexes with red-shifted absorptions that possess long-lived, redox-active excited states that are useful for various applications, including solar energy conversion and photochemotherapy.

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 µm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 µm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

Visible-Light-Driven Photosystems Using Heteroleptic Cu(I) Photosensitizers and Rh(III) Catalysts To Produce H2.

The synthesis of two new heteroleptic Cu(I) photosensitizers (PS), [Cu(Xantphos)(NN)]PF6 (NN = biq = 2,2'-biquinoline, dmebiq = 2,2'-biquinoline-4,4'-dimethyl ester; Xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene), along with the associated structural, photophysical, and electrochemical properties, are described. The biquinoline diimine ligand extends the PS light absorbing properties into the visible with a maximum absorption at 455 and 505 nm for NN = biq and dmebiq, respectively, in CH2Cl2 solvent. Following photoexcitation, both Cu(I) PS are emissive at low energy, albeit displaying stark differences in their excited state lifetimes (τMLCT = 410 ± 5 (biq) and 44 ± 4 ns (dmebiq)). Cyclic voltammetry indicates a Cu-based HOMO and NN-based LUMO for both complexes, whereby the methyl ester substituents stabilize the LUMO within [Cu(Xantphos)(dmebiq)]+ by ∼0.37 V compared to the unsubstituted analogue. When combined with H2O, N,N-dimethylaniline (DMA) electron donor, and cis-[Rh(NN)2Cl2]PF6 (NN = Me2bpy = 4,4'-dimethyl-2,2'-bipyridine, bpy = 2,2'-bipyridine, dmebpy = 2,2'-bipyridine-4,4'-dimethyl ester) water reduction catalysts (WRC), photocatalytic H2 evolution is only observed using the [Cu(Xantphos)(biq)]+ PS. Furthermore, the choice of cis-[Rh(NN)2Cl2]+ WRC strongly affects the catalytic activity with turnover numbers (TONRh = mol H2 per mol Rh catalyst) of 25 ± 3, 22 ± 1, and 43 ± 3 for NN = Me2bpy, bpy, and dmebpy, respectively. This work illustrates how ligand modification to carefully tune the PS light absorbing, excited state, and redox-active properties, along with the WRC redox potentials, can have a profound impact on the photoinduced intermolecular electron transfer between components and the subsequent catalytic activity.

The contribution of alu elements to mutagenic DNA double-strand break repair.

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

New Rh2(II,II) Complexes for Solar Energy Applications: Panchromatic Absorption and Excited-State Reactivity.

The new heteroleptic paddlewheel complexes cis-[Rh2(µ-form)2(µ-np)2][BF4]2, where form = p-ditolylformamidinate (DTolF) or p-difluorobenzylformamidinate (F-form) and np = 1,8-napthyridyine, and cis-Rh2(µ-form)2(µ-npCOO)2 (npCOO- = 1,8-naphthyridine-2-carboxylate), were synthesized and characterized. The complexes absorb strongly throughout the ultraviolet (λmax = 300 nm, ε = 20 300 M-1 cm-1) and visible regions (λmax = 640 nm ε = 3500 M-1 cm-1), making them potentially useful new dyes with panchromatic light absorption for solar energy conversion applications. Ultrafast and nanosecond transient absorption and time-resolved infrared spectroscopies were used to characterize the identity and dynamics of the excited states, where singlet and triplet Rh2/form-to-naphthyridine, metal/ligand-to-ligand charge-transfer (ML-LCT) excited states were observed in all four complexes. The npCOO- complexes exhibit red-shifted absorption profiles extending into the near-IR and undergo photoinitiated electron transfer to generate reduced methyl viologen, a species that persists in the presence of a sacrificial donor. The energy of the triplet excited state of each complex was estimated from energy-transfer quenching experiments using a series of organic triplet donors (E(3ππ*) from 1.83 to 0.78 eV). The singlet reduction (+0.6 V vs Ag/AgCl) potentials, and singlet and triplet oxidation potentials (-1.1 and -0.5 V vs Ag/AgCl, respectively) were determined. Based on the excited-state lifetimes and redox properties, these complexes represent a new class of light absorbers with potential application as dyes for charge injection into semiconductor solar cells and in sensitizer-catalyst assemblies for photocatalysis that operate with irradiation from the ultraviolet to ∼800 nm.

Judicious Ligand Design in Ruthenium Polypyridyl CO2 Reduction Catalysts to Enhance Reactivity by Steric and Electronic Effects.

A series of RuII polypyridyl complexes of the structural design [RuII (R-tpy)(NN)(CH3 CN)]2+ (R-tpy=2,2':6',2''-terpyridine (R=H) or 4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine (R=tBu); NN=2,2'-bipyridine with methyl substituents in various positions) have been synthesized and analyzed for their ability to function as electrocatalysts for the reduction of CO2 to CO. Detailed electrochemical analyses establish how substitutions at different ring positions of the bipyridine and terpyridine ligands can have profound electronic and, even more importantly, steric effects that determine the complexes' reactivities. Whereas electron-donating groups para to the heteroatoms exhibit the expected electronic effect, with an increase in turnover frequencies at increased overpotential, the introduction of a methyl group at the ortho position of NN imposes drastic steric effects. Two complexes, [RuII (tpy)(6-mbpy)(CH3 CN)]2+ (trans-[3]2+ ; 6-mbpy=6-methyl-2,2'-bipyridine) and [RuII (tBu-tpy)(6-mbpy)(CH3 CN)]2+ (trans-[4]2+ ), in which the methyl group of the 6-mbpy ligand is trans to the CH3 CN ligand, show electrocatalytic CO2 reduction at a previously unreactive oxidation state of the complex. This low overpotential pathway follows an ECE mechanism (electron transfer-chemical reaction-electron transfer), and is a direct result of steric interactions that facilitate CH3 CN ligand dissociation, CO2 coordination, and ultimately catalytic turnover at the first reduction potential of the complexes. All experimental observations are rigorously corroborated by DFT calculations.

Activating a Low Overpotential CO2 Reduction Mechanism by a Strategic Ligand Modification on a Ruthenium Polypyridyl Catalyst.

The introduction of a simple methyl substituent on the bipyridine ligand of [Ru(tBu3 tpy)(bpy)(NCCH3 )](2+) (tBu3 tpy=4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine; bpy=2,2'-bipyridine) gives rise to a highly active electrocatalyst for the reduction of CO2 to CO. The methyl group enables CO2 binding already at the one-electron reduced state of the complex to enter a previously not accessible catalytic cycle that operates at the potential of the first reduction. The complex turns over with a Faradaic efficiency close to unity and at an overpotential that is amongst the lowest ever reported for homogenous CO2 reduction catalysts.

New Rh2(II,II) Architecture for the Catalytic Reduction of H⁺.

Formamidinate-bridged Rh2(II,II) complexes containing diimine ligands of the formula cis-[Rh2(II,II)(µ-DTolF)2(NN)2](2+) (Rh2-NN2), where DTolF = p-ditolylformamidinate and NN = dppn (benzo[i]dipyrido[3,2-a:2',3'-h]quinoxaline), dppz (dipyrido[3,2-a:2',3'-c]phenazine), and phen (1,10-phenanthroline), electrocatalytically reduce H(+) to H2 in DMF solutions containing CH3COOH at a glassy carbon electrode. Cathodic scans in the absence of acid display a Rh(III,II/II,II) reduction at -0.90 V vs Fc(+)/Fc followed by NN(0/-) reduction at -1.13, -1.36, and -1.65 V for Rh2-dppn2, Rh2-dppz2, and Rh2-phen2, respectively. Upon the addition of acid, Rh2-dppn2 and Rh2-dppz2 undergo reduction-protonation-reduction at each pyrazine-containing NN ligand prior to the Rh2(II,II/II,I) reduction. The Rh2(II,I) species is then protonated at one of the metal centers, resulting in the formation of the corresponding Rh2(II,III)-hydride. In the case of Rh2-phen2, the reduction of the phen ligand is followed by intramolecular electron transfer to the Rh2(II,II) core in the presence of protons to form a Rh2(II,III)-hydride species. Further reduction and protonation at the Rh2 core for all three complexes rapidly catalyzes H2 formation with varied calculated turnover frequencies (TOF) and overpotential values (η): 2.6 × 10(4) s(-1) and 0.56 V for Rh2-dppn, 2.8 × 10(4) s(-1) and 0.50 V for Rh2-dppz2, and 5.9 × 10(4) s(-1) and 0.64 V for Rh2-phen2. Bulk electrolysis confirmed H2 formation, and further CH3COOH addition regenerates H2 production, attesting to the robust nature of the architecture. The cis-[Rh2(II,II)(µ-DTolF)2(NN)2](2+) architecture benefits by combining electron-rich formamidinate bridges, a redox-active Rh2(II,II) core, and electron-accepting NN diimine ligands to allow for the electrocatalysis of H(+) substrate to H2 fuel.

Nonchromophoric halide ligand variation in polyazine-bridged Ru(II),Rh(III) bimetallic supramolecules offering new insight into photocatalytic hydrogen production from water.

The new bimetallic complex [(Ph2phen)2Ru(dpp)RhBr2(Ph2phen)](PF6)3 (1) (Ph2phen = 4,7-diphenyl-1,10-phenanthroline; dpp = 2,3-bis(2-pyridyl)pyrazine) was synthesized and characterized to compare with the Cl(-) analogue [(Ph2phen)2Ru(dpp)RhCl2(Ph2phen)](PF6)3 (2) in an effort to better understand the role of halide coordination at the Rh metal center in solar H2 production schemes. Electrochemical properties of complex 1 display a reversible Ru(II/III) oxidation, and cathodic scans indicate multiple electrochemical mechanisms exist to reduce Rh(III) by two electrons to Rh(I) followed by a quasi-reversible dpp(0/-) ligand reduction. The weaker σ-donating ability of Br(-) vs Cl(-) impacts the cathodic electrochemistry and provides insight into photocatalytic function by these bimetallic supramolecules. Complexes 1 and 2 exhibit identical light-absorbing properties with UV absorption dominated by intraligand (IL) π → π* transitions and visible absorption by metal-to-ligand charge transfer (MLCT) transitions to include a lowest energy Ru(dπ) → dpp(π*) (1)MLCT transition (λ(abs) = 514 nm; ε = 16 000 M(-1) cm(-1)). The relatively short-lived, weakly emissive Ru(dπ) → dpp(π*) (3)MLCT excited state (τ = 46 ns) for both bimetallic complexes is attributed to intramolecular electron transfer from the (3)MLCT excited state to populate a low-energy Ru(dπ) → Rh(dσ*) triplet metal-to-metal charge transfer ((3)MMCT) excited state that allows photoinitiated electron collection. Complex 1 outperforms the related Cl(-) bimetallic analogue 2 as a H2 photocatalyst despite identical light-absorbing and excited-state properties. Additional H2 experiments with added halide suggest ion pairing plays a role in catalyst deactivation and provides new insight into observed differences in H2 production upon halide variation in Ru(II),Rh(III) supramolecular architectures.

Tuning the Photophysical Properties of Ru(II) Monometallic and Ru(II),Rh(III) Bimetallic Supramolecular Complexes by Selective Ligand Deuteration.

A series of three new complexes of the design [(TL)2Ru(BL)](2+), two new complexes of the design [(TL)2Ru(BL)Ru(TL)2](4+), and three new complexes of the design [(TL)2Ru(BL)RhCl2(TL)](3+) (TL = bpy or d8-bpy; BL = dpp or d10-dpp; TL = terminal ligand; BL = bridging ligand; bpy = 2,2'-bipyridine; dpp = 2,3-bis(2-pyridyl)pyrazine) were synthesized and the (1)H NMR spectroscopy, electrochemistry, electronic absorbance spectroscopy, and photophysical properties studied. Incorporation of deuterated ligands into the molecular architecture simplifies the (1)H NMR spectra, allowing for complete (1)H assignment of [(d8-bpy)2Ru(dpp)](PF6)2 and partial assignment of [(bpy)2Ru(d10-dpp)](PF6)2. The electrochemistry for the deuterated and nondeuterated species showed nearly identical redox properties. Electronic absorption spectroscopy of the deuterated and nondeuterated complexes are superimposable with the lowest energy transition being Ru(dπ) → BL(π*) charge transfer in nature (BL = dpp or d10-dpp). Ligand deuteration impacts the excited-state properties with an observed increase in the quantum yield of emission (Φ(em)) and excited-state lifetime (τ) of the Ru(dπ) → d10-dpp(π*) triplet metal-to-ligand charge transfer ((3)MLCT) excited state when dpp is deuterated, and a decrease in the rate constant for nonradiative decay (knr). Choice of ligand deuteration between bpy and dpp strongly impacts the observed photophysical properties with BL = d10-dpp complexes showing an enhanced Φ(em) and τ, providing further support that the lowest electronic excited state populated via UV or visible excitation is the photoactive Ru(dπ) → dpp(π*) CT excited state. The Ru(II),Rh(III) complex incorporating the deuterated BL shows increased hydrogen production compared to the variants incorporating the protiated BL, while demonstrating identical dynamic quenching behaviors in the presence of sacrificial electron donor.

The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms.

Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3' end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications.

Mechanistic insight into the electronic influences imposed by substituent variation in polyazine-bridged ruthenium(II)/rhodium(III) supramolecules.

Unusual and unprecedented multipathway electrochemical mechanisms for a new class of supramolecular Ru/Rh bimetallic photocatalysts have been uncovered. The near isoenergetic Rh(dσ*) and bridging ligand(π*) molecular orbitals and a rate of halide loss that occurs on the cyclic voltammetry timescale provide a series of closely related complexes which display four different electrochemical mechanisms. A single complex displays two concurrent electrochemical pathways in marked contrast to all previously studied cis-[Rh(NN)(2)X(2)] motifs.

Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.

Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), which predisposes to tumorigenesis. To interrogate this risk, we developed a two- color flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of cells following a DSB. After this initial increase, cells with LOH decrease due to a competitive disadvantage with parental cells, but if isolated, they stably propagate. Segmental loss from terminal deletions with de novo telomere addition and nonreciprocal translocations is observed as well as whole chromosome loss, especially following a centromeric DSB. LOH spans megabases distal from the DSB, but also frequently tens of megabases centromere-proximal. Inhibition of microhomology-mediated end joining massively increases LOH, which is synergistically increased with concomitant inhibition of canonical nonhomologous end joining. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end joining inhibition.

Molecular basis of FIGNL1 in dissociating RAD51 from DNA and chromatin.

Maintaining genome integrity is an essential and challenging process. RAD51 recombinase, the central player of several crucial processes in repairing and protecting genome integrity, forms filaments on DNA. RAD51 filaments are tightly regulated. One of these regulators is FIGNL1, that prevents persistent RAD51 foci post-damage and genotoxic chromatin association in cells. The cryogenic electron microscopy structure of FIGNL1 in complex with RAD51 reveals that the FIGNL1 forms a non-planar hexamer and RAD51 N-terminus is enclosed in the FIGNL1 hexamer pore. Mutations in pore loop or catalytic residues of FIGNL1 render it defective in filament disassembly and are lethal in mouse embryonic stem cells. Our study reveals a unique mechanism for removing RAD51 from DNA and provides the molecular basis for FIGNL1 in maintaining genome stability.

Subunit variation to uncover properties of polyazine-bridged Ru(II), Pt(II) supramolecules with low lying charge separated states providing insight into the functioning as H2O reduction photocatalysts to produce H2.

Two new structurally diverse polyazine-bridged Ru(II),Pt(II) tetrametallic complexes, [{(Ph2phen)2Ru(dpp)}2Ru(dpp)PtCl2](PF6)6 (1a) and [{(Ph2phen)2Ru(dpp)}2Ru(dpq)PtCl2](PF6)6 (2a) (Ph2phen = 4,7-diphenyl-1,10-phenanthroline, dpp = 2,3-bis(2-pyridyl)pyrazine, dpq = 2,3-bis(2-pyridyl)quinoxaline), as well as their trimetallic precursors have been synthesized to provide a comparison for detailed analysis to elucidate component effects in the previously reported photocatalyst [{(phen)2Ru(dpp)}2Ru(dpq)PtCl2](PF6)6 (4a) (phen = 1,10-phenanthroline). Electrochemistry shows terminal Ru based highest occupied molecular orbitals (HOMOs) with remote BL' (BL' = bridging ligand coupling central Ru and cis-PtCl2 moiety) based lowest unoccupied molecular orbitals (LUMOs). Population of a lowest-lying charge separated ((3)CS) excited state with oxidized terminal Ru and reduced remote BL' via intramolecular electron transfer is predicted by electrochemical analysis and is observed through steady-state and time-resolved emission studies as well as emission excitation profiles which display unusual nonunity population of the lowest lying emissive Ru→dpp (3)MLCT (metal-to-ligand charge transfer) state. Each tetrametallic complex is an active photocatalyst for H2 production from H2O with 2a showing the highest activity (94 TON (turnover number) in 10 h, where TON = mol H2/mol catalyst). The nature of the bridging ligand coupling the trimetallic light absorber to the cis-PtCl2 moiety has a significant impact on the catalyst activity, correlated to the degree of population of the (3)CS excited state. The choice of terminal ligand affects visible light absorption and has a minor influence on photocatalytic H2 production from H2O. Evidence that an intact supramolecule functions as the photocatalyst includes a strong dependence of the photocatalysis on the identity of BL', an insensitivity to Hg(l), no detectable H2 production from the systems with the trimetallic synthons and cis-[PtCl2(DMSO)2] as well as spectroscopic analysis of the photocatalytic system.

Total wash elimination for solid phase peptide synthesis.

We present a process for solid phase peptide synthesis (SPPS) that completely eliminates all solvent intensive washing steps during each amino acid addition cycle. A key breakthrough is the removal of a volatile Fmoc deprotection base through bulk evaporation at elevated temperature while preventing condensation on the vessel surfaces with a directed headspace gas flushing. This process was demonstrated at both research and production scales without any impact on product quality and when applied to a variety of challenging sequences (up to 89 amino acids in length). The overall result is an extremely fast, high purity, scalable process with a massive waste reduction (up to 95%) while only requiring 10-15% of the standard amount of base used. This transformation of SPPS represents a step-change in peptide manufacturing process efficiency, and should encourage expanded access to peptide-based therapeutics.

Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation.

Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3' end of the intron RNA to the 5' end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5' junctions with 5' exon resections, 5' intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.