A Shock-Tube Study of the Rate Constant of PH3 + M ⇄ PH2 + H + M (M = Ar) Using PH3 Laser Absorption.

Phosphine (PH3) is a highly reactive and toxic gas. Prior experimental investigations of PH3 pyrolysis reactions have included only low-temperature measurements. This study reports the first shock-tube measurements of PH3 pyrolysis using a new PH3 laser absorption technique near 4.56 µm. Experiments were conducted in mixtures of 0.5% PH3/Ar behind reflected shock waves at temperatures of 1460-2013 K and pressures of ∼1.3 and ∼0.5 atm. The PH3 time histories displayed two-stage behavior similar to that previously observed for NH3 decomposition, suggesting by analogy that the rate constant for PH3 + M ⇄ PH2 + H + M (R1) could be determined. A simple three-step mechanism was assembled for data analysis. In a detailed kinetic analysis of the first-stage PH3 decomposition, values of k1,0 were obtained and best described by (in cm3·mol-1·s-1) k1,0 = 7.78 × 1017 exp(-80,400/RT), with units of cal, mol, K, s, and cm3. Agreement between the 1.3 and 0.5 atm data confirmed that the measured k1,0 was in the low-pressure limit. Agreement of the experimental k1,0 with ab initio estimates resolved the question of the main pathway of PH3 decomposition: it proceeds as PH3 ⇄ PH2 + H instead of PH3 ⇄ PH + H2.

DNA expansions generated by human Polµ on iterative sequences.

Polµ is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polµ is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polµ-mediated dinucleotide expansion as a function of the sequence context. In this model, Polµ requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5'-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polµ residues His(329) and Arg(387) as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polµ at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.

DNA-binding determinants promoting NHEJ by human Polµ.

Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair.

Influence of excitation wavelength (UV or visible light) on the photocatalytic activity of titania containing gold nanoparticles for the generation of hydrogen or oxygen from water.

Gold nanoparticles supported on P25 titania (Au/TiO(2)) exhibit photocatalytic activity for UV and visible light (532 nm laser or polychromatic light λ > 400 nm) water splitting. The efficiency and operating mechanism are different depending on whether excitation occurs on the titania semiconductor (gold acting as electron buffer and site for gas generation) or on the surface plasmon band of gold (photoinjection of electrons from gold onto the titania conduction band and less oxidizing electron hole potential of about -1.14 V). For the novel visible light photoactivity of Au/TiO(2), it has been determined that gold loading, particle size and calcination temperature play a role in the photocatalytic activity, the most active material (Φ(H2) = 7.5% and Φ(O2) = 5.0% at 560 nm) being the catalyst containing 0.2 wt % gold with 1.87 nm average particle size and calcined at 200 °C.

Gold catalyzes the Sonogashira coupling reaction without the requirement of palladium impurities.

By a combination of kinetic and theoretical studies it is concluded that gold is intrinsically active to perform the Sonogashira coupling reaction between phenylacetylene and iodobenzene. The presence of Pd impurities is not mandatory for catalyst activity.

Ceria nanoparticles as heterogeneous catalyst for CO2 fixation by omega-aminoalcohols.

In contrast to gamma-Al(2)O(3), TiO(2), ZrO(2), MgO and Y(2)O(3), CeO(2) is a reusable catalyst for the reaction of CO(2) with omega-aminoalcohols to form cyclic carbamates; the highest yield (68%) was obtained for the preparation of N-alkyl 1,3-oxazolidin-2-ones from N-alkyl ethanolamines.

Limited terminal transferase in human DNA polymerase mu defines the required balance between accuracy and efficiency in NHEJ.

DNA polymerase mu (Polmu) is a family X member implicated in DNA repair, with template-directed and terminal transferase (template-independent) activities. It has been proposed that the terminal transferase activity of Polmu can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polmu, we have identified a specific DNA ligand residue (Arg(387)) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polmu mutant R387K (mimicking TdT) displayed a large increase in terminal transferase activity, but a weakened interaction with ssDNA. That paradox can be explained by the regulatory role of Arg(387) in the translocation of the primer from a non-productive E:DNA complex to a productive E:DNA:dNTP complex in the absence of a templating base, which is probably the rate limiting step during template-independent synthesis. Further, we show that the Polmu switch from terminal transferase to templated insertions in NHEJ reactions is triggered by recognition of a 5'-P at a second DNA end, whose 3'-protrusion could provide a templating base to facilitate such a special "pre-catalytic translocation step." These studies shed light on the mechanism by which a rate-limited terminal transferase activity in Polmu could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.

Structure of a NHEJ polymerase-mediated DNA synaptic complex.

Nonhomologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway required to maintain genome stability. Many prokaryotes possess a minimalist NHEJ apparatus required to repair DSBs during stationary phase, composed of two conserved core proteins, Ku and ligase D (LigD). The crystal structure of Mycobacterium tuberculosis polymerase domain of LigD mediating the synapsis of two noncomplementary DNA ends revealed a variety of interactions, including microhomology base pairing, mismatched and flipped-out bases, and 3' termini forming hairpin-like ends. Biochemical and biophysical studies confirmed that polymerase-induced end synapsis also occurs in solution. We propose that this DNA synaptic structure reflects an intermediate bridging stage of the NHEJ process, before end processing and ligation, with both the polymerase and the DNA sequence playing pivotal roles in determining the sequential order of synapsis and remodeling before end joining.

Structure and function of a mycobacterial NHEJ DNA repair polymerase.

Non homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks in prokaryotes requires Ku and a specific multidomain DNA ligase (LigD). We present crystal structures of the primase/polymerisation domain (PolDom) of Mycobacterium tuberculosis LigD, alone and complexed with nucleotides. The PolDom structure combines the general fold of the archaeo-eukaryotic primase (AEP) superfamily with additional loops and domains that together form a deep cleft on the surface, likely used for DNA binding. Enzymatic analysis indicates that the PolDom of LigD, even in the absence of accessory domains and Ku proteins, has the potential to recognise DNA end-joining intermediates. Strikingly, one of the main signals for the specific and efficient binding of PolDom to DNA is the presence of a 5'-phosphate group, located at the single/double-stranded junction at both gapped and 3'-protruding DNA molecules. Although structurally unrelated, Pol lambda and Pol mu, the two eukaryotic DNA polymerases involved in NHEJ, are endowed with a similar capacity to bind a 5'-phosphate group. Other properties that are beneficial for NHEJ, such as the ability to generate template distortions and realignments of the primer, displayed by Pol lambda and Pol mu, are shared by the PolDom of bacterial LigD. In addition, PolDom can perform non-mutagenic translesion synthesis on termini containing modified bases. Significantly, ribonucleotide insertion appears to be a recurrent theme associated with NHEJ, maximised in this case by the deployment of a dedicated primase, although its in vivo relevance is unknown.

A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis.

Human DNA polymerase mu (Polmu) is a family X member that has terminal transferase activity but, in spite of a non-orthodox selection of the template information, displays its maximal catalytic efficiency in DNA-templated reactions. As terminal deoxynucleotidyl transferase (TdT), Polmu has a specific loop (loop1) that could provide this enzyme with its terminal transferase activity. When loop1 was deleted, human Polmu lacked TdT activity but improved DNA-binding and DNA template-dependent polymerization. Interestingly, when loop1 from TdT was inserted in Polmu (substituting its cognate loop1), the resulting chimaera displayed TdT activity, preferentially inserting dGTP residues, but had a strongly reduced template-dependent polymerization activity. Therefore, a specialized loop in Polmu, that could adopt alternative conformations, appears to provide this enzyme with a dual capacity: (i) template independency to create new DNA information, in which loop1 would have an active role by acting as a 'pseudotemplate'; (ii) template-dependent polymerization, in which loop1 must allow binding of the template strand. Recent in vivo and in vitro data suggest that such a dual capacity could be advantageous to resolve microhomology-mediated end-joining reactions.

A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining.

Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe.

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3'-->5' proofreading activity and its preferred substrates are small gaps with a 5'-phosphate group. Similarly to Polmu, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1-2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polmu, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polmu. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polbeta and Pollambda, and unlike Polmu, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polbeta, Pollambda and Polmu.

Lack of sugar discrimination by human Pol mu requires a single glycine residue.

DNA polymerase mu (Pol mu) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol mu is not able to discriminate against the 2'-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol mu is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol mu based on the available Pol beta and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol mu was mutated to the consensus tyrosine present in Pol beta, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol mu in DNA repair.

Are there mutator polymerases?

DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

DNA polymerase lambda, a novel DNA repair enzyme in human cells.

DNA polymerase lambda (pol lambda) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in pol lambda supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol lambda to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol lambda inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5'-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol lambda is a novel beta-like DNA polymerase. However, the high affinity of pol lambda for dNTPs (37-fold over pol beta) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol beta and pol lambda have nonredundant in vivo functions.