Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

Interplay Between Side Chain Pattern,Polymer Aggregation,and Charge Carrier Dynamics in PBDTTPD:PCBM Bulk‐Heterojunction Solar Cells

Poly(benzo[1,2‐b:4,5‐b′]dithiophene–alt–thieno[3,4‐c]pyrrole‐4,6‐dione) (PBDTTPD) polymer donors with linear side‐chains yield bulk‐heterojunction (BHJ) solar cell power conversion efficiencies (PCEs) of about 4% with phenyl‐C₇₁‐butyric acid methyl ester (PC₇₁BM) as the acceptor, while a PBDTTPD polymer with a combination of branched and linear substituents yields a doubling of the PCE to 8%. Using transient optical spectroscopy it is shown that while the exciton dissociation and ultrafast charge generation steps are not strongly affected by the side chain modifications, the polymer with branched side chains exhibits a decreased rate of nongeminate recombination and a lower fraction of sub‐nanosecond geminate recombination. In turn the yield of long‐lived charge carriers increases, resulting in a 33% increase in short circuit current (J _sc). In parallel, the two polymers show distinct grazing incidence X‐ray scattering spectra indicative of the presence of stacks with different orientation patterns in optimized thin‐film BHJ devices. Independent of the packing pattern the spectroscopic data also reveals the existence of polymer aggregates in the pristine polymer films as well as in both blends which trap excitons and hinder their dissociation.     

In vitro poly(ADP‐ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP‐ribosyl)ated in vitro. The modifying product, poly(ADP‐ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis‐specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP‐ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP‐ribose)polymerase in the presence of [³²P] NAD. In parallel, poly(ADP‐ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [³²P] NAD. In both experiments, the poly(ADP‐ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP‐ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10–12 units long, whereas longer chains (≤20 ADP‐R units) were linked to H1t. Individual poly(ADP‐ribosyl)ated H1s were complexed with homologous H1‐depleted oligonucleosomes (0.5–2.5 kbp) in order to measure their ability to condensate chromatin, in comparison with the native ones. Circular dichroism showed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA‐binding properties of the analyzed H1s. In particular, the poly(ADP‐ribosyl)ated H1a and H1t had opposite effects on the condensation of H1‐depleted oligonucleosomes. J. Cell. Biochem. 76:20–29, 1999. © 1999 Wiley‐Liss, Inc.     

Structural basis for tankyrase‐RNF146 interaction reveals noncanonical tankyrase‐binding motifs

Poly(ADP‐ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase‐1 and ‐2; a.k.a. PARP‐5a and ‐5b) is involved in mitosis, telomere length regulation, GLUT‐4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase‐binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C‐terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate‐binding region of TNKS, forming a robust one‐to‐one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide‐binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase‐binding proteins including those of IF4A1 and NELFE.     

Deficiency of terminal ADP‐ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Poly(ADP-ribose)polymerase: a perplexing participant in cellular responses to DNA breakage

Poly(ADP-ribose) polymerase is a major nuclear protein of 116 kd, coded by a gene on chromosome 1, that plays a role in cellular responses to DNA breakage. The polymerase binds to DNA at single- and double-strand breaks and synthesizes long branched chains of poly(ADP-ribose), which covalently, but transiently, modifies itself and numerous other cellular proteins and depletes cells of NAD+. This much is known, but the physiological role of the polymerization-degradation cycle is still unclear. Poly(ADP-ribosyl)ation of proteins generally inhibits their function and can dissociated chromatin proteins from DNA. Inhibition of poly(ADP-ribose) polymerase increases to toxicity of alkylating agents and some other DNA-damaging agents and increases sister-chromatid exchange frequencies. During repair of alkylation damage, inhibition of poly(ADP-ribose) polymerase makes no change in excision of damaged products. increases the total number of repair patches, accelerates the rejoining of DNA breaks, and makes variable increases or decreases in net break frequencies. The polymerization cycle consequently is a major player in the response of cells to DNA breakage, but the game it plays is yet to be explained.     

Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR–protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.     

The effect of in vivo treatment with triiodothyronine on the in vitro synthesis of protein-poly(ADP)-ribose adducts by isolated cardiocyte nuclei and the separation of poly(ADP)-ribosylated proteins by phenol extraction and electrophoresis

In vivo treatment of rats with triiodothyronine (30 micrograms/100 g of body weight for 4 consecutive days) inhibited poly(ADP)-ribose polymerase activity of cardiocyte nuclei, but low enzymatic activity of nuclei of noncardiocyte origin remained unaffected. RNA synthesis in cardiocyte nuclei isolated from triiodothyronine-treated rats was augmented. A positive correlation was observed between the degree of inhibition of poly(ADP)-ribose polymerase and cardiac ventricular enlargement in triiodothyronine-treated animals. RNA synthesis in isolated cardiocyte nuclei was inhibited by in vitro poly(ADP)-ribosylation only when cardiocyte nuclei were obtained from triiodothyronine-treated animals. In vitro poly(ADP)-ribosylated proteins were isolated from cardiocyte nuclei by solvent partitioning between phenol and aqueous phases. About 90% of the protein-poly(ADP)-ribose adducts partitioned into the aqueous fraction, and the chain length of polymers in this phase was between medium (n = 4-9) and long (n greater than 32), whereas the phenol phase contained protein-oligomer and monomer adducts. Not only the chain length of oligomers but the nature of modified proteins appeared to participate in determining the partitioning of polymer-protein adducts, and different proteins were separated from the two phases by gel electrophoresis. More than 90% of protein-polymer adducts formed by cardiocyte nuclei were not extracted by 0.25 N HCl, indicating prevalence of nonhistone proteins as polymer acceptors. Gel electrophoresis and near quantitative recovery of adducts in a gel system that protected from degradation of adducts to free polymers confirmed the predominance of nonhistone proteins as main acceptors and demonstrated an artifact of autoradiography that seemed to indicate histone H1 as a significant acceptor. Treatment with triiodothyronine diminished poly(ADP)-ribosylation of certain groups of proteins more than others, implying some degree of selectivity of action of the hormone. Catabolism of the polymer in vitro was not affected by triiodothyronine treatment.     

Poly(2-aminoethyl methacrylate) with well-defined chain length for DNA vaccine delivery to dendritic cells

Poly(2-aminoethyl methacrylate) (PAEM) homopolymers with defined chain length and narrow molecular weight distribution were synthesized using atom transfer radical polymerization (ATRP), and a comprehensive study was conducted to evaluate the colloidal properties of PAEM/plasmid DNA polyplexes, the uptake and subcellular trafficking of polyplexes in antigen-presenting dendritic cells (DCs), and the biological performance of PAEM as a potential DNA vaccine carrier. PAEM of different chain length (45, 75, and 150 repeating units) showed varying strength in condensing plasmid DNA into narrowly dispersed nanoparticles with very low cytotoxicity. Longer polymer chain length resulted in higher levels of overall cellular uptake and nuclear uptake of plasmid DNA, but shorter polymer chains favored intracellular and intranuclear release of free plasmid from the polyplexes. Despite its simple chemical structure, PAEM transfected DCs very efficiently in vitro in media with or without serum and led to phenotypic maturation of DCs. When a model antigen-encoding ovalbumin plasmid was used, transfected DCs stimulated the activation of na?ve CD8(+) T cells to produce high levels of interferon-γ. The efficiency of transfection, DC maturation, and CD8(+) T cell activation showed varying degrees of polymer chain-length dependence. These structurally defined cationic polymers may have much potential as efficient DNA vaccine carriers and immunostimulatory adjuvants. They may also serve as a model material system for elucidating structural and intracellular mechanisms of polymer-mediated DNA vaccine delivery.     

Patterns of LPS synthesis in gram negative bacteria

Lipopolysaccharide (LPS), a lipid based carbohydrate polymer, is found in the outer membrane of Gram negative bacteria where it plays a vital role in its structure and function. It is chiefly responsible for the toxic effects of the bacterial diseases caused by these organisms and plays a role in the organisms defense against host immune attack. In recent experiments using high resolution gel techniques Goldman & Leive (1980) have revealed an unexpected heterogeneity in the distribution of polymer lengths found in the membrane with lengths ranging from 0 (lipid-A-core) to nearly 40 sugar units. Monomer units are an essential element of the synthesis process. Working with mutant strains of E. coli and Salmonella typhimurium, they have also shown that these bacteria will continue to synthesize LPS molecules with very long chains even though monomer unit production is severely reduced. The steps involved in the synthesis of LPS are known and in this paper it is shown that the results of Goldman & Leive cannot be obtained assuming the synthesis process is length independent as has been suggested. Moreover, the paradoxical persistence of long chains in spite of monomer suppression has a simple explanation once length dependence is assumed. These conclusions result from the analysis of a Markov chain model of synthesis.     

Structural insights into SAM domain‐mediated tankyrase oligomerization

Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP‐ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain‐mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head‐to‐tail polymer that facilitates TNKS self‐association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM‐TNKS2 SAM) hetero‐oligomeric structures mediated by their SAM domains. Though wild‐type tankyrase proteins have very low solubility, model‐based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP‐ribosyl)ation (PARylation) and PARylation‐dependent ubiquitylation.     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Structural characterization and buffering capacity in relation to the transfection efficiency of biodegradable polyurethane

Inefficient release of polymer/DNA complexes from endocytic vesicles into the cytoplasm and the cytotoxic nature of cationic polymers are two of the primary causes of poor gene delivery. EG-polyurethane [poly(ethylene glycol)-PU, Poly 1], EGDM-polyurethane [poly(ethylene glycol), 2-(dimethylamino)ethylamine-PU, Poly 2], and MDEADM-polyurethane [N-methyldiethanolamine, 2-(dimethylamino)ethylamine-PU, Poly 3] were designed in this study to overcome these obstacles. The structural characteristics of polyurethanes and physicochemical properties of their formed complexes with DNA were determined to correlate their transfection efficiency. The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than those of Poly 2 and Poly 1. This study reveals that the amine groups in the polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Also the size and binding properties of DNA and polymeric transfectants can be in correlation to the transfection efficiency of resulting DNA/polymer complexes.     

Molecular Dynamics Study of Water in Hydrogels

Abstract

Molecular dynamics simulations are performed for aqueous solutions of polymers: Poly (vinyl alcohol) (PVA), Poly (vinyl methylether) (PVME), and Poly (N-isopropyl acrylamide) (PNiPAM). The distributions and dynamics of hydrogen-bonds, the translational diffusion of water, and the orientational relaxation of water are analyzed to investigate the properties of water which is highly influenced by the surrounding polymer chains. The water molecules around the polymer chains are highly hindered by the chains.     

Poly(ADP-ribose) catabolism in mammalian cells

Poly(ADP-ribose) catabolism is a complex situation involving many proteins and DNA. We have developed anin vitro turnover system where poly(ADP-ribose) metabolism is monitored in presence of different relative amounts of two principal enzymes poly(ADP-ribose) transferase and poly(ADP-ribose) glycohydrolase along with other proteins and DNA. Our current results reviewed here show that the quality of polymer, i.e. chain length and complexity, as well as preference for the nuclear substrate varies depending upon the availability of poly(ADP-ribose) glycohydrolase. These results are interpreted in the light of the recent data implicating poly(ADP-ribose) metabolism in DNA-repair. (Mol Cell Biochem 138: 45–52 1994)     

Conformational effects and cooperative interactions in poly[5(6)-vinylbenzimidazole]-catalyzed solvolyses of anionic,long-chain substrates

A new synthetic procedure for the monomer, 5(6)-vinylbenzimidazole, has been developed. Also, a new method for the formation of the benzimidazole moiety is reported. The monomer, 5(6)-vinylbenzimidazole, was subjected to a free radical solution polymerization and a solid state thermal polymerization. Poly[5(6)-vinyl-benzimidazole], prepared by the solution-free radical polymerization, was shown to be a linear addition polymer with pendant benzimidazole groups, whereas the thermally prepared polymer was shown to have a different structure. The solvolyses of negatively charged esters with varying aliphatic chain lengths catalyzed by poly[5(6)-vinylbenzimidazole] and compared to monomeric benzimidazole in 40% 1-propanol-water at 26°C revealed that the polymer was more efficient and that its activity was a function of the degree of ionization. Neutral-neutral and neutral-anionic benzimidazole interactions are suggested. The polymer conformation was found to have a significant effect on the solvolysis reactions.