Lipase-catalyzed polymerization of lactones and linear hydroxyesters

Ring-opening polymerization of six lactones and condensation polymerization of six linear hydroxyesters were carried out at 45°C using a lipase from Pseudomonas sp. Ring-opening polymerization of the lactones gave both higher molecular weight and higher monomer conversion than condensation of corresponding linear hydroxyesters. Lipase-catalyzed copolymerization of -caprolactone with lactide and cyclopentadecanolide gave higher molecular weight compared with copolymerization of -caprolactone with corresponding linear hydroxyesters. © Rapid Science Ltd. 1998     

Electronic structure and reaction mechanism as guides to the design of hybrid inorganic-organic polymers

Systematic approaches to the design of organofunctional cyclophosphazene monomers and their conversion to organic backbone polymers with cyclophosphazene substituents are described. A variety of methods including DFT calculations, photoelectron spectroscopy and copolymerization reactivity ratios show that the π electrons in an olefin attached to a cyclophosphazene undergo σ polarization towards the phosphazene. This effect may be mediated by electron donors on the olefin or, more effectively, by an insulating function between the olefin and the inorganic ring. Two optimized monomers, P₃N₃Cl₅OR (RC₆H₄CHCH₂, (CH₂)₄OC (O)CMeCH₂), have been prepared and their homo- and copolymerization behavior explored. Mixed phosphazene substituent systems have also been developed. Per substituted monomers, P₃N₃(OR)₆, have been developed and converted to cross linked, hyperbranched and cyclomatrix materials via multi arm polymerization.     

The role of weak interactions in lignin polymerization

Lignin is the most abundant natural polymer composed by aromatic moieties. Its chemical composition and its abundance have focused efforts to unlock its potential as a source of aromatic compounds for many years. The lack of a proper way for lignin de-polymerization has hampered its success as a natural solution for commodity aromatic chemicals, which is also due to the lack of understanding of the underlying mechanisms of lignin polymerization. A fuller fundamental understanding of polymerization mechanisms could lead to improvements in de-polymerization strategies, and therefore a proper methodology and a predictive theoretical framework are required for such purpose. This work presents a complete computational study on some of the key steps of lignin polymerization mechanisms. Density functional theory (DFT) calculations have been performed to evaluate the most appropriate methodology and to compute the chemical structures and reaction enthalpies for the monolignol dimerization, the simplest key step that controls the polymerization. Quantum theory of atoms in molecules (QTAIM) has been applied to understand the coupling reaction mechanisms, for which the radical species and transition states (TSs) involved have been characterized. The coupling that leads to the formation of the β–O–4 linkage has been theoretically reproduced according to proposed mechanisms, for which weak interactions have been found to play a key role in the arrangement of reactants. The hydrogen bond formed between the oxygen of the phenoxy radical, and the alcohol of the aliphatic chain, together with the interaction between aromatic rings, locates the reactants in a position that favors such β–O–4 linkage.

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Graphical Abstract QTAIM analysis of the complex between coumaryl and coniferyl alcohols. It emphasizes the importance of weak interactions during the formation of beta-O-4 linkages in the polymerization of lignin.

     

Finger Domain Mutation Affects Enzyme Activity,DNA Replication Efficiency,and Fidelity of an Exonuclease-Deficient DNA Polymerase of Herpes Simplex Virus Type 1

The catalytic subunit of herpes simplex virus DNA polymerase (Pol), a member of the B family polymerases, possesses both polymerase and exonuclease activities. We previously demonstrated that a recombinant virus (YD12) containing a double mutation within conserved exonuclease motif III of the Pol was highly mutagenic and rapidly evolved to contain an additional leucine-to-phenylalanine mutation at residue 774 (L774F), which is located within the finger subdomain of the polymerase domain. We further demonstrated that the recombinant L774F virus replicated DNA with increased fidelity and that the L774F mutant Pol exhibited altered enzyme kinetics and impaired polymerase activity to extension from mismatched primer termini. In this study, we demonstrated that addition of the L774F mutation to the YD12 Pol did not restore the exonuclease deficiency. However, the polymerase activity of the YD12 Pol to extension from mismatched primer termini and on the nucleotide incorporation pattern was altered upon addition of the L774F mutation. The L774F mutation-containing YD12 Pol also supported the growth of viral progeny and replicated DNA more efficiently and more accurately than did the YD12 Pol. Together, these studies demonstrate that a herpes simplex virus Pol mutant with a highly mutagenic ability can rapidly acquire additional mutations, which may be selected for their survival and outgrowth. Furthermore, the studies demonstrate that the polymerase activity of HSV-1 Pol on primer extension is influenced by sequence context and that herpes simplex virus type 1 Pol may dissociate more frequently at G·C sites during the polymerization reaction. The implications of the findings are discussed.Herpes simplex virus (HSV) DNA polymerase consists of the catalytic subunit of the polymerase (Pol) and the processivity factor UL42. The Pol subunit contains three well-defined activities: polymerization (replication), exonuclease proofreading (editing), and UL42 binding (5, 6, 28). The UL42 binding activity is mediated by amino acid residues located at the C terminus (5, 6). Although the UL42 binding residues are unique to certain alphaherpesvirus DNA polymerases, the sequences comprising the polymerase and exonuclease domains are conserved among the B family (or the α-like) polymerases (2-4). The exonuclease domain of the HSV type 1 (HSV-1) Pol contains conserved exonuclease I (Exo I), II, and III motifs, whereas the polymerase domain contains seven conserved regions (I to VII); conserved region IV overlaps with the Exo II motif. The Exo III motif is located within the δ region C, which is highly conserved among the B family polymerases (Fig. ​(Fig.1A).1A). These conserved regions are located within the palm, the thumb, and the finger subdomains, which comprise the structural components of the polymerase domain. The crystal structure of the HSV-1 Pol subunit revealed three grooves that form the putative polymerase, exonuclease, and DNA binding sites. The putative exonuclease site is defined as a groove formed between the exonuclease domain and the tip of the thumb subdomain. The palm and thumb subdomains form a groove proposed to be the putative duplex DNA binding site for both the editing and the polymerization complexes (23). Thus, the polymerase and exonuclease domains of HSV-1 are structurally and functionally interconnected (1, 7, 16, 21, 23, 27, 28), although they are organized into two different domains.Open in a separate windowFIG. 1.(A) Schematic diagram of the conserved regions and motifs within HSV-1 Pol. The relative locations of the conserved regions of HSV-1 Pol are shown at the top; regions I to VII and δ region C are represented by open boxes. The conserved exonuclease motifs I, II, and III are indicated with closed boxes. The functional and structural domains (determined by crystal structure analysis [23]) of the HSV-1 Pol are shown below. The N-terminal domain (N domain) is composed of two regions separated by the 3′-to-5′ exonuclease domain (23). (B) Schematic diagram of wild-type and mutant YDL Pol. The BamHI fragment of the wild-type pol from the plasmid pHC629 is shown at the top. The relative location of conserved region VI and the Exo III motif are shown below, with corresponding wild-type and mutant (YDL) amino acid sequences. B, BamHI; M, MstI; N, NotI.The high fidelity of DNA replication is achieved by three different mechanisms: nucleotide discrimination during the polymerization reaction, editing immediately after the polymerization reaction, and postreplication repair. HSV-1 mutant Pol containing mutations within the conserved regions of the polymerase domain can result in altered enzyme kinetics and DNA replication fidelity (8, 9, 11, 12, 18, 26). Similarly, mutation of conserved Exo domain residues can lead to the loss of exonuclease activity and to altered nucleotide selection and incorporation kinetics as well as the mutator phenotype (1, 10, 13, 14, 21, 25). Our previous studies demonstrated that a mutant Pol (YD12) containing a tyrosine-to-histidine substitution at residue 577 and an aspartic acid-to-alanine substitution at residue 581 (Y577H/D581A) is exonuclease deficient (exo⁻) and that recombinant virus expressing the mutant Pol exhibits a mutator phenotype in vivo (14). However, this recombinant virus rapidly evolved to contain an additional leucine-to-phenylalanine substitution at residue 774 (L774F), which is located within conserved region VI of the polymerase domain (18). Interestingly, a recombinant virus containing the L774F Pol mutation exhibits increased fidelity of DNA replication (18). Our recent study also demonstrated that the mutant L774F Pol exhibits altered enzyme kinetics (26). These results led to the hypothesis that the emerged L774F mutation in the context of the YD12 Pol mutant may also affect enzyme activity, DNA replication, and fidelity.     

New polyazole derivatives from 2-(2,4-dichlorophenyl)-1,3-dioxolane. Antifungal activity. Structure-activity relationships

Six new polyazole derivatives from 2-(2,4-dichlorophenyl)-1,3-dioxolane were studied for their antifungal activity against pathogenic fungi for humans and animals. The antimycotic effect was largely restricted to the filamentous fungi with Aspergillus fumigatus and Scedosporium apiospermum being remarkably susceptible. Three compounds were as effective as the two references, ketoconazole and oxiconazole. The structure-activity relationships revealed that an oxime group combined with four chlorine atoms increased the antifungal properties of the chemical series.This revised version was published online in October 2005 with corrections to the Cover Date.     

On the dynamics of DNA in aqueous solution. Pulse radiolysis studies using the light scattering detection method

Summary The kinetics of DNA chain breakage in solution induced by 2 µs pulses of 15 MeV electrons were investigated by light scattering. On irradiating native calf thymus DNA at room temperature the decrease of light scattering intensity (LSI) - due to double strand ruptures - shows a fast decay with a half life _1/2 of about 30 ms as well as a slow decay with _1/2 of about 10 s. With increasing temperature (20–40° C) both the total degree of degradation and the fraction of the fast decay increase due to the facilitated melting of segments between two single strand breaks on alternate strands forming a double strand break. Above 40° C a third mode of LSI decay with _1/2 of 5–10 s arises, indicating detachment of relatively long segments.The total relative decrease of LSI after irradiation A, which can be taken as a measure of the degree of degradation, follows the square of the absorbed dose in the case of native DNA, whereas on irradiating denatured DNA A rises linearly with dose. The decay of LSI due to the degradation of denatured DNA is much faster than that of native DNA with _1/2 down to 150 µs, depending on the absorbed dose. The half lives are interpreted in terms of the separation of fragments by diffusion and of the melting of double strand segments between two single strand breaks.     

Human DNA Polymerase η Is Required for Common Fragile Site Stability during Unperturbed DNA Replication

Human DNA polymerase η (Pol η) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Pol η in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol η from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Pol η-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol ι, the closest relative of Pol η. Our results identify a new role for Pol η in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Pol η in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage.Mutations in the POLH gene that encodes DNA polymerase η (Pol η) are responsible for the variant form of xeroderma pigmentosum (XP-V). XP-V is a rare autosomal recessive disorder characterized by extreme sensitivity to sunlight and a very high incidence of sunlight-induced skin cancer, as are the other forms of “classical” XP (17, 27). However, in contrast to the other nucleotide excision repair (NER)-defective XP complementation groups (XP-A to XP-G), XP-V cells have normal NER but cannot support translesion synthesis (TLS) past DNA-containing cyclobutane pyrimidine dimers (CPDs) (27). Purified Pol η, the TLS polymerase that is mutated in XP-V, is able to synthesize past this lesion with a high level of efficiency (28), and in a majority of cases it inserts the correct nucleotide, adenine, opposite the two thymines contained in the cyclobutane pyrimidine dimer ring (26).The ability to replicate efficiently past UV pyrimidine dimers has been the principal—or sole—function assigned thus far to Pol η. In the absence of Pol η, cells display an increased rate of UV-induced mutagenesis and carcinogenesis (23) that may reflect inefficient or error-prone synthesis by another polymerase. In mouse cells, this back-up polymerase may be Pol ι (12). Despite its ability to replicate past cyclobutane pyrimidine dimers, Pol η does not appear to be able to carry out TLS past the other major UV photoproduct, the pyrimidine (6-4) pyrimidone photoproduct [(6-4)PP] in vitro or in vivo. It can, however, replicate past a limited number of other types of DNA damage in vitro, albeit with a lower level of efficiency than past CPDs (21). Whether the bypass of these lesions is performed in vivo by Pol η is less clear. For example, XP-V cells are sensitive to cisplatin, suggesting that bypass of cisplatin lesions may depend on Pol η (1). Combined NER- and Pol η-mediated lesion bypass has also been suggested as the likely mechanism for repairing DNA interstrand cross-links formed by mitomycin C (46) and psoralen (32). In contrast, Pol η does not appear to play a role in replication past endogenous lesions such as 8-oxoguanine (3) or abasic sites (2).It has been difficult to visualize or identify sites of action of Pol η or any of the other TLS polymerases by immunofluorescence due to their low levels of expression. However, in cells that mildly overexpress Pol η, it has been possible to localize the polymerase to nuclear replication factories during S phase. This localization depends on several motifs located close to the C terminus of Pol η, including an NLS and a ubiquitin-binding zinc finger domain (7, 18). Localization of Pol η in replication factories may concentrate the polymerase near sites of replication to facilitate recruitment to carry out TLS. If cells cannot remove or synthesize through a lesion blocking the replication fork, then homology-dependent recombinational repair (HRR) may be used to restart the replication fork (11, 34). RAD51-mediated HRR has been shown to be important for the repair of DNA damage during replication in all organisms (20, 31, 42). Recent evidence has suggested that Pol η, in addition to its role in TLS, may participate in HRR. This has been suggested by analyses of gene conversion in chicken DT40 cells during immunoglobulin gene diversification (19), as well as by in vitro experiments showing that Pol η is capable of promoting extension of the invading strand in D-loop structures to facilitate RAD52-mediated second-end capture during recombination-mediated repair (29, 30). The functional importance of this observation is less clear. Recent evidence from yeast argues that the bulk of heteroduplex DNA strand extension during HRR is mediated by the preferential recruitment of a replicative DNA polymerase, Pol δ (25). Moreover, there is no obvious recombination deficit in XP-V patients or in XP-V cells beyond a modest elevation in the frequency of UV-induced sister chromatid exchanges (10).In order to better understand the functional roles and importance of Pol η in human cells, we used short hairpin RNAs (shRNAs) to selectively deplete Pol η from cells and then determined how the loss of Pol η affected cell cycle progression, DNA replication dynamics, and cell proliferation in otherwise unperturbed cells. These experiments revealed an unexpected role for Pol η in maintaining chromosomal stability and preventing common fragile site (CFS) breakage during unperturbed S phase. Our results thus broaden the functional role of Pol η in human cells to include the maintenance of genomic stability during unperturbed DNA replication in S phase.     

Half-sandwich Mo(III) complexes with asymmetric diazadiene ligands

The asymmetric 1,4-diazadiene ligands R^∗NCHCHNR^∗ [R^∗ = (S)-CH(CH₃)Ph], , and 2,2′-bis(4-ethyloxazoline), as-ox, have been used to generate half-sandwich Mo^III derivatives by addition to Cp₂Mo₂Cl₄. Ligand affords a mononuclear, paramagnetic 17-electron product, , whereas as-ox leads to the isolation of a dinuclear compound where only one molecule of ligand has been added per two Mo atoms, Cp₂Mo₂Cl₄(as-ox). In the presence of free as-ox, this compound coexists with the paramagnetic mononuclear complex in solution. Both products are capable of controlling the radical polymerization of styrene under typical atom transfer radical polymerization (ATRP) conditions. However, the tacticity of the resulting polystyrene does not differ from that given by conventional free radical polymerization.