DNA lesion bypass polymerases open up

Structures of catalytic fragments of two DNA lesion bypass DNA polymerases, yeast DNA polymerase eta and an archeon DinB homolog, have recently been solved. These structures share several common architectural and structural features observed in other DNA polymerases, including a hand-like architecture with fingers, palm, and thumb subdomains. The new structures provide the first structural insights into DNA lesion bypass. The fingers and thumb are smaller than those in other DNA polymerases. Modeled substrates suggest that the fingers in the vicinity of the incoming nucleotide is closed, a conformation not previously observed for an unliganded polymerase. However, the template binding pocket appears to be more open, indicating that for DNA polymerase eta, a covalently linked thymine-thymine dimer could be accommodated.     

One channel: open and closed

Structural information about the prokaryotic KirBac3.1 inward rectifier family K(+) channel from Magnetospirillum magnetotacticum is reported. These results from two-dimensional electron cryomicroscopy (EM) shed light on the gating mechanism of members of the Kir channel family.     

An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases.

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions. In combination with the scintillation proximity assay (SPA[trade]), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides. A proof of the concept is obtained using pseudo-thymidine (psiT), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psiT arising from the carbon-carbon bond between the sugar and the base make it an interesting probe for the importance of conformational restraints in the active site of polymerases during primer elongation. From a pool of commercially available thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides containing psiT. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psiT are presented. This is the first time that PCR has been performed with a C-nucleoside.     

Selection for litter size in pigs. II. Efficiency of closed and open selection lines*

A selection experiment on litter size in the pig was carried on for seventeen generations in an Inra experimental herd. The founder population was made up of 10 males and 120 females from the Large White breed. Selection was first performed for ten generations in a closed line, compared to a control line derived from the same founder population. Selection was carried on within sire family on the total number of piglets born in the first two litters of the dam (TB1 + TB2). After ten generations, the selection criterion became dam TB1 only. The control line was then discontinued and a panel of frozen semen from the 11th generation boars was created for later comparisons. The selected line was opened to gilt daughters of hyperprolific boars and sows, at a rate of 1/8 per generation, and the same selection procedure was applied irrespective of the origin of the gilt. During the whole experiment, the number of ova shed (OS) and the number of live embryos (LE) at 30 days in the 3rd pregnancy were recorded. These two parts of the experiment were analysed using REML estimation of genetic parameters and a BLUP-Animal Model in order to estimate the responses to selection. Significant heritabilities for TB1, TB2, OS and LE were obtained, i.e. 0.10, 0.05, 0.43 and 0.19, respectively. Significant common environment variances and covariances were estimated for nearly all traits. Significantly positive BLUP responses per generation were observed from G0 to G17 for TB1 (+0.086), TB2 (+0.078), OS (+0.197) and LE (+0.157). However, the responses were 3- to 4-fold higher in the G12–G17 interval compared to G0–G11, and they were also in fair agreement with previous estimates based on standard least-squares procedures, using the control line and the control frozen semen panel. Since G11, the selection intensity was increased by nearly 80 p. cent compared to the previous generations, and the proportion of hyperprolific ancestry increased up to 65 p. cent in the sows of the last generation. The total genetic gain of about 1.4 piglets at birth per litter could be shared between a gain due to immigration, of about 0.8 piglets per litter, and a within-line selection gain of about 0.6 piglets. Thus by combining selection and immigration in the second part of the experiment, advantage could be taken from both the genetic superiority of the immigrants and the higher internal selection intensity made possible by immigration.     

KcsA closed and open: modelling and simulation studies

Bacterial homologues of mammalian potassium channels provide structures of two states of a gated K channel. Thus, the crystal structure of KcsA represents a closed state whilst that of MthK represents an open state. Using homology modelling and molecular dynamics simulations we have built a model of the transmembrane domain of KcsA in an open state and have compared its conformational stability with that of the same domain of KcsA in a closed state. Approximate Born energy calculations of monovalent cations within the two KcsA channel states suggest that the intracellular hydrophobic gate in the closed state provides a barrier of height ~5 kT to ion permeation, whilst in the open state the barrier is absent. Simulations (10 ns duration) in an octane slab (a simple membrane mimetic) suggest that closed- and open-state models are of comparable conformational stability, both exhibiting conformational drifts of ~3.3 Å C RMSD relative to the respective starting models. Substantial conformational fluctuations are observed in the intracellular gate region during both simulations (closed state and open state). In the simulation of open-state KcsA, rapid (<5 ns) exit of all three K⁺ ions occurs through the intracellular mouth of the channel. Helix kink and swivel motion is observed at the molecular hinge formed by residue G99 of the M2 helix. This motion is more substantial for the open- than for the closed-state model of the channel.     

Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations

Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

An inhibitor of DNA polymerases alpha and delta in calf thymus DNA.

Most, although not all, samples of commercial calf thymus DNA were strongly inhibitory to DNA polymerase alpha; the inhibition made the DNA useless as a template for this enzyme. In a pre-assembled DNA polymerase assay mixture (minus enzyme but including activated DNA) the inhibition tended to diminish with time but at a rate that was not predictable, and some inhibition usually persisted. It was concluded that the inhibition was the result of contamination of the DNA by a heparin-like material on the basis of the following: 1) the inhibition could be reversed by treatment of the DNA with heparinase; 2) both the endogenous inhibitory effect of calf thymus DNA as well as the inhibitory effect of heparin on DNA polymerase alpha are reversed by protamine (which is known to prevent the antithrombin activity of heparin); 3) both the endogenous inhibition and inhibition by heparin are also reversed by ampholyte (which also prevents the antithrombin activity of heparin); and 4) both the endogenous and the heparin-induced inhibitory effects display the same spectrum of activity against mammalian DNA polymerases, i.e. both DNA polymerases alpha and delta are extremely sensitive whereas, DNA polymerases beta and gamma are resistant. The last result also suggests the use of heparin as a specific inhibitor of purified mammalian DNA polymerases alpha and delta, similar to the use of aphidicolin.     

Mild cycles open closed communities to ecological restoration

Species loss is a global issue. With up to a million species at risk and insufficient protected area to maintain the world's biodiversity, humanity will increasingly need to rely on species re‐introductions to locally restore diversity and function. However, such restoration attempts are bound to fail when ecological communities get locked in a closed state that is resistant to recovery. It is presently unknown how to repair these closed systems. We use mathematical models to identify ways out of this problem. We first show how ecological communities may enter a closed state, to then explain how to open them up again for restoration of their original diversity. We find that restoration is often still possible shortly after initial species loss, as (1) the secondary extinctions that produce closure have not happened yet and (2) mild population fluctuations still allow successful repair during a transient postdisturbance phase. However, after this typically short window of opportunity for restoration, the system enters a new equilibrium, which may be a closed state. Our analysis shows how to take ecological communities out of the closed state: Appropriate management of carrying capacities produces a regime of mild population fluctuations that opens a window for successful species re‐introductions. These windows can be perpetually recurring or permanently open. Such opportunities for repair can be absent under regimes of wild cycles or perfect stability. We conclude that mild cycles may open windows of opportunity for the repair of communities that have become resistant to recovery.