Tailor-made polymer tracers reveal the role of clay minerals on colloidal transport in carbonate media.

HYPOTHESIS: Host rock weathering and incipient pedogenesis result in the exposition of minerals, e.g., clay minerals in sedimentary limestones. Once exposed, these minerals provide the surfaces for fluid-solid interactions that control the fate of dissolved or suspended compounds such as organic matter and colloids. However, the functional and compositional diversity of organic matter and colloids limits the assessment of reactivity and availability of clay mineral interfaces. Such assessment demands a mobile compound with strong affinity to clay surfaces that is alien to the subsurface. EXPERIMENT: We approached this challenge by using poly(ethylene glycol) (PEG) as interfacial tracer in limestone weathering experiments. FINDINGS: PEG adsorption and transport was dependent on the availability of clay mineral surfaces and carbonate dissolution dynamics. In addition, PEG adsorption featured adsorption-desorption hysteresis which retained PEG mass on clay mineral surfaces. This resulted in different PEG transport for experiments conducted consecutively in the same porous medium. As such, PEG transport was reconstructed with a continuum-scale model parametrized by a Langmuir-type isotherm including hysteresis. Thus, we quantified the influence of exposed clay mineral surfaces on the transport of organic colloids in carbonate media. This renders PEG a suitable model colloid tracer for the assessment of clay surface exposition in porous media.

Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance.

Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation (LLPS) were studied in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of dispersed hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and its interaction with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. Addition of RNA to dispersed hnRNP A1 induced liquid-droplet formation. Such LLPS depended on RNA concentration and sequence, with continuous wave EPR spectroscopy showing an influence of RNA point mutations on local protein dynamics. We propose that an interplay of sequence-specific RNA binding and LLPS contributes to regulation of specific RNA segregation during stress response.

Characterization of Weak Protein Domain Structure by Spin-Label Distance Distributions.

Function of intrinsically disordered proteins may depend on deviation of their conformational ensemble from that of a random coil. Such deviation may be hard to characterize and quantify, if it is weak. We explored the potential of distance distributions between spin labels, as they can be measured by electron paramagnetic resonance techniques, for aiding such characterization. On the example of the intrinsically disordered N-terminal domain 1-267 of fused in sarcoma (FUS) we examined what such distance distributions can and cannot reveal on the random-coil reference state. On the example of the glycine-rich domain 188-320 of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) we studied whether deviation from a random-coil ensemble can be robustly detected with 19 distance distribution restraints. We discuss limitations imposed by ill-posedness of the conversion of primary data to distance distributions and propose overlap of distance distributions as a fit criterion that can tackle this problem. For testing consistency and size sufficiency of the restraint set, we propose jack-knife resampling. At current desktop computers, our approach is expected to be viable for domains up to 150 residues and for between 10 and 50 distance distribution restraints.

Rpb4 subunit functions mainly in mRNA synthesis by RNA polymerase II.

RNA polymerase II (Pol II) is the central enzyme that carries out eukaryotic mRNA transcription and consists of a 10-subunit catalytic core and a subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to dissociate from Pol II, enter the cytoplasm, and function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, as well as evidence arguing against an important cytoplasmic role in mRNA degradation. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis to show that Rpb4 deletion in Saccharomyces cerevisiae causes a drastic defect in mRNA synthesis that is compensated by down-regulation of mRNA degradation, resulting in mRNA level buffering. Deletion of Rpb4 can be rescued by covalent fusion of Rpb4 to the Pol II core subunit Rpb2, which largely restores mRNA synthesis and degradation defects caused by Rpb4 deletion. Thus, Rpb4 is a bona fide Pol II core subunit that functions mainly in mRNA synthesis.

Evolution of two modes of intrinsic RNA polymerase transcript cleavage.

During gene transcription, the RNA polymerase (Pol) active center can catalyze RNA cleavage. This intrinsic cleavage activity is strong for Pol I and Pol III but very weak for Pol II. The reason for this difference is unclear because the active centers of the polymerases are virtually identical. Here we show that Pol II gains strong cleavage activity when the C-terminal zinc ribbon domain (C-ribbon) of subunit Rpb9 is replaced by its counterpart from the Pol III subunit C11. X-ray analysis shows that the C-ribbon has detached from its site on the Pol II surface and is mobile. Mutagenesis indicates that the C-ribbon transiently inserts into the Pol II pore to complement the active center. This mechanism is also used by transcription factor IIS, a factor that can bind Pol II and induce strong RNA cleavage. Together with published data, our results indicate that Pol I and Pol III contain catalytic C-ribbons that complement the active center, whereas Pol II contains a non-catalytic C-ribbon that is immobilized on the enzyme surface. Evolution of the Pol II system may have rendered mRNA transcript cleavage controllable by the dissociable factor transcription factor IIS to enable promoter-proximal gene regulation and elaborate 3'-processing and transcription termination.

Roles of Hop1 and Mek1 in meiotic chromosome pairing and recombination partner choice in Schizosaccharomyces pombe.

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.

The heme oxygenase 1 product biliverdin interferes with hepatitis C virus replication by increasing antiviral interferon response.

UNLABELLED: The anti-inflammatory and antiapoptotic heme degrading enzyme heme oxygenase-1 (HO-1) has been shown recently to interfere with replication of hepatitis C virus (HCV). We investigated the effect of HO-1 products carbon monoxide (CO), iron and biliverdin on HCV replication using the replicon cell lines Huh-5-15 and LucUbiNeo-ET, stably expressing HCV proteins NS3 through NS5B. Incubation of these cell lines in the presence of the CO donor methylene chloride transiently reduced HCV replication, whereas an increase of iron in cell culture by administration of FeCl(3) or iron-saturated lactoferrin did not interfere with HCV replication. Likewise, depletion of iron by deferoxamine during induction of HO-1 by cobalt-protoporphyrin IX did not restore HCV replication. The most prominent effect was observed after incubation of replicon cell lines in the presence of biliverdin. Biliverdin seems to interfere with HCV replication-mediated oxidative stress by inducing expression of antiviral interferons, such as interferon alpha2 and alpha17. CONCLUSION: The antioxidant biliverdin reduces HCV replication in vitro by triggering the antiviral interferon response and might improve HCV therapy in the future.

Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.

We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3' end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3' nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.

Structure-function studies of the RNA polymerase II elongation complex.

RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin alpha-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.

Sites of strong Rec12/Spo11 binding in the fission yeast genome are associated with meiotic recombination and with centromeres.

Meiotic recombination arises from Rec12/Spo11-dependent formation of DNA double-strand breaks (DSBs) and their subsequent repair. We identified Rec12-binding peaks across the Schizosaccharomyces pombe genome using chromatin immunoprecipitation after reversible formaldehyde cross-linking combined with whole-genome DNA microarrays. Strong Rec12 binding coincided with previously identified DSBs at the recombination hotspots ura4A, mbs1, and mbs2 and correlated with DSB formation at a new site. In addition, Rec12 binding corresponded to eight novel conversion hotspots and correlated with crossover density in segments of chromosome I. Notably, Rec12 binding inversely correlated with guanine-cytosine (GC) content, contrary to findings in Saccharomyces cerevisiae. Although both replication origins and Rec12-binding sites preferred AT-rich gene-free regions, they seemed to exclude each other. We also uncovered a connection between binding sites of Rec12 and meiotic cohesin Rec8. Rec12-binding peaks lay often within 2.5 kb of a Rec8-binding peak. Rec12 binding showed preference for large intergenic regions and was found to bind preferentially near to genes expressed strongly in meiosis. Surprisingly, Rec12 binding was also detected in centromeric core regions, which raises the intriguing possibility that Rec12 plays additional roles in meiotic chromosome dynamics.

Single-molecule tracking of mRNA exiting from RNA polymerase II.

Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5' end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5'-capping enzyme and other RNA processing factors.

Molecular basis of RNA-dependent RNA polymerase II activity.

RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene transcription. There is, however, evidence that Pol II also possesses RNA-dependent RNA polymerase (RdRP) activity. Pol II can use a homopolymeric RNA template, can extend RNA by several nucleotides in the absence of DNA, and has been implicated in the replication of the RNA genomes of hepatitis delta virus (HDV) and plant viroids. Here we show the intrinsic RdRP activity of Pol II with only pure polymerase, an RNA template-product scaffold and nucleoside triphosphates (NTPs). Crystallography reveals the template-product duplex in the site occupied by the DNA-RNA hybrid during transcription. RdRP activity resides at the active site used during transcription, but it is slower and less processive than DNA-dependent activity. RdRP activity is also obtained with part of the HDV antigenome. The complex of transcription factor IIS (TFIIS) with Pol II can cleave one HDV strand, create a reactive stem-loop in the hybrid site, and extend the new RNA 3' end. Short RNA stem-loops with a 5' extension suffice for activity, but their growth to a critical length apparently impairs processivity. The RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes.

DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes.

Chromatin-remodeling complexes can translocate nucleosomes along the DNA in an ATP-coupled reaction. This process is an important regulator of all DNA-dependent processes because it determines whether certain DNA sequences are found in regions between nucleosomes with increased accessibility for other factors or wrapped around the histone octamer complex. In a comparison of seven different chromatin-remodeling machines (ACF, ISWI, Snf2H, Chd1, Mi-2, Brg1, and NURF), it is demonstrated that these complexes can read out DNA sequence features to establish specific nucleosome-positioning patterns. For one of the remodelers, ACF, we identified a 40-bp DNA sequence element that directs nucleosome positioning. Furthermore, we show that nucleosome positioning by the remodelers ACF and Chd1 is determined by a reduced affinity to the end product of the translocation reaction. The results suggest that the linkage of differential remodeling activities with the intrinsic binding preferences of nucleosomes can result in establishing distinct chromatin structures that depend on the DNA sequence and define the DNA accessibility for other protein factors.

Multisubunit RNA polymerases melt only a single DNA base pair downstream of the active site.

To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.

Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast.

Two rounds of chromosome segregation after only a single round of DNA replication enable the production of haploid gametes from diploid precursors during meiosis. To identify genes involved in meiotic chromosome segregation, we developed an efficient strategy to knock out genes in the fission yeast on a large scale. We used this technique to delete 180 functionally uncharacterized genes whose expression is upregulated during meiosis. Deletion of two genes, sgo1 and mde2, caused massive chromosome missegregation. sgo1 is required for retention of centromeric sister-chromatid cohesion after anaphase I. We show here that mde2 is required for formation of the double-strand breaks necessary for meiotic recombination.

The meiotic recombination hot spot ura4A in Schizosaccharomyces pombe.

The meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the ura4+ gene 15 kb centromere-proximal to ade6 on chromosome III. Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion. This report concerns the characterization of 12 spontaneous ura4A mutants. A gradient of conversion ranging from 18% at the 5' end to 6% at the 3' end was detected. A novel phenomenon also was discovered: a mating-type-related bias of conversion. The allele entering with the h+ parent acts preferentially as the acceptor for conversion (ratio of 3:2). Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5' end, and more often than not covers the entire gene. Restoration repair of markers at the 5' end was inferred. Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded recombinant frequencies higher than the sum of the frequencies measured in the subintervals. Finally, marker effects on recombination were defined for two of the ura4A mutations.

Different frameshift mutation spectra in non-repetitive DNA of MutSalpha- and MutLalpha-deficient fission yeast cells.

A frameshift reversion assay has been established for Schizosaccharomyces pombe, which allows detection of deletions and insertions of nucleotides in a non-repetitive DNA sequence. Compared to wild type, frameshift mutation rates were increased in the mismatch repair (MMR) mutants msh2, msh6, mlh1, and pms1, but not in a swi4 strain (defective in the Msh3 homologue). Rates were also elevated in the DNA nuclease-deficient strains rad2 (defective in the FEN-1 homologue) and exo1. In MutSalpha-deficient strains, msh2 and msh6, most of the reversions were 1bp deletions. In contrast, mlh1 and pms1 mutants, defective in MutLalpha, accumulated significantly more 2bp insertions, preferentially of the type CG to (CG)(2). Such duplications were less frequent in double mutants additionally defective in msh2, msh6, rad2, or exo1. Thus, accumulation of (CG)(2) in MutLalpha-deficient strains depends on the presence of MutSalpha, Rad2 and Exo1.