Exonuclease Degradation Research Articles

Dynamic changes of telomeric restriction fragment (TRF) lengths in cells during the developmental process from embryos to seedlings and a comparison with the embryonal calli in Ginkgo biloba L.

Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location of Arabidopsis-type TTTAGGG tandem repeats were thought to be highly conserved. The terminal location of Ginkgo biloba L. consisting of TTTAGGG tandem repeats, were confirmed by Bal31 exonuclease degradation and Southern blotting. By comparing telomeric restriction fragment (TRF) lengths at different developmental stages from embryos to seedlings, a fluctuant tendency towards variation was found in these samples. The TRF length of embryos was also compared with that of embryonal calli and an upward trend was discovered in callus culture. The results suggest that there should be a telomerase mechanism or/and ALT mechanism for the maintenance of telomere length.

Is a Thymine Dimer Replicated via a Transient Abasic Site Intermediate? A Comparative Study Using Non-Natural Nucleotides

UV light causes the formation of thymine dimers that can be misreplicated to induce mutagenesis and carcinogenesis. This report describes the use of a series of non-natural indolyl nucleotides in probing the ability of the high-fidelity bacteriophage T4 DNA polymerase to replicate this class of DNA lesion. Kinetic data reveal that indolyl analogues containing large pi-electron surface areas are incorporated opposite the thymine dimer almost as effectively as an abasic site, a noninstructional lesion. However, there are notable differences in the kinetic parameters for each DNA lesion that indicate distinct mechanisms for their replication. For example, the rate constants for incorporation opposite a thymine dimer are considerably slower than those measured opposite an abasic site. In addition, the magnitude of these rate constants depends equally upon contributions from pi-electron density and the overall size of the analogue. In contrast, binding of a nucleotide opposite a thymine dimer is directly correlated with the overall pi-electron surface area of the incoming dXTP. In addition to defining the kinetics of polymerization, we also provide the first reported characterization of the enzymatic removal of natural and non-natural nucleotides paired opposite a thymine dimer through exonuclease degradation or pyrophosphorolysis activity. Surprisingly, the exonuclease activity of the bacteriophage enzyme is activated by a thymine dimer but not by an abasic site. This dichotomy suggests that the polymerase can "sense" bulky lesions to partition the damaged DNA into the exonuclease domain. The data for both nucleotide incorporation and excision are used to propose models accounting for polymerase "switching" during translesion DNA synthesis.

Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway

Most common point mutations occurring spontaneously or induced by ionizing radiation are C → T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5′ to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2′-deoxycytidine (5ohC) and alpha-anomeric 2′-deoxycytidine (αdC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (NΔ31, NΔ35 and NΔ61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3′ → 5′ exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3′ → 5′ exonuclease activities generate a single-strand gap at the 5′ side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo.

In Vitro Inhibition of Promyelocytic Leukemia/Retinoic Acid Receptor-α (PML/RARα) Expression and Leukemogenic Activity by DNA/LNA Chimeric Antisense Oligos

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

3′–5′ exonuclease activity of human apurinic/apyrimidinic endonuclease 1 towards DNAs containing dNMP and their modified analogs at the 3′ end of single strand DNA break

Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.

Nucleotide-Dependent Degradation of Nucleic Acids by DNA and RNA Polymerases

In this review we summarize the current knowledge about recently discovered reactions of nucleotide-dependent nucleic acid degradation catalyzed by DNA and RNA polymerases. These reactions consist in the excision of the 3'-nucleotide of nascent DNA or RNA chain in the presence of non-complementary r/dNTPs. In the case of DNA polymerases the dinucleoside-5',5"-tetraphosphate as a product of the reaction is formed. In contrast, in the case of RNA polymerases non-complementary r/dNTPs stimulate 3'-->5' exonuclease degradation of RNA transcript. The possible role of these reactions in fidelity of DNA and RNA synthesis, in resistance of HIV reverse transcriptase towards nucleoside inhibitors is discussed.

Regulation of Unsaturated Fatty Acid Biosynthesis in Saccharomyces: THE ENDOPLASMIC RETICULUM MEMBRANE PROTEIN, Mga2p, A TRANSCRIPTION ACTIVATOR OF THE OLE1 GENE, REGULATES THE STABILITY OF THE OLE1 mRNA THROUGH EXOSOME-MEDIATED MECHANISMS

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta9 fatty-acid desaturase, whose expression is regulated through transcriptional and mRNA stability controls. In wild type cells grown on fatty acid-free medium, OLE1 mRNA has a half-life of 10 +/- 1.5 min (basal stability) that becomes highly unstable when cells are exposed to unsaturated fatty acids (regulated stability). Activation of OLE1 transcription is dependent on N-terminal fragments of two membrane proteins, Mga2p and Spt23p, that are proteolytically released from the membrane by a ubiquitin-mediated mechanism. Surprisingly, disruption of the MGA2 gene also reduces the half-life of the OLE1 transcript and abolishes fatty acid regulated instability. Disruption of its cognate, SPT23, has no effect on the half-life of the mRNA. Mga2p appears to have two distinct functions with respect to the OLE1 mRNA stability: a stabilizing effect in cells grown in fatty acid-free medium and a destabilizing function in cells that are exposed to unsaturated fatty acids. These functions are independent of OLE1 transcription and can confer basal and regulated stability on OLE1 mRNAs that are produced under the control of the unrelated GAL1 promoter. Expression of soluble, N-terminal fragments of Mga2p stabilize the transcript but do not confer fatty acid-regulated instability on the mRNA suggesting that the stabilizing functions of Mga2p do not require membrane processing and that modifications to the protein introduced during proteolysis may play a role in the destabilizing effect. An analysis of mutants that are defective in mRNA degradation indicate that the Mga2p-requiring control mechanism that regulates the fatty acid-mediated instability of the OLE1 transcript acts by activating exosomal 3' --> 5'-exonuclease degradation activity.

Single step high-throughput determination of Toll-like receptor 4 polymorphisms

Background: Toll-like receptors are central components of host defence in humans, responsible for recognition of pathogen-associated molecular patterns and activation of innate immunity. Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS) and other microbial components, thereby initiating the expression and release of pro-inflammatory cytokines. The common, frequently co-segregating allelic variants Asp299Gly and Thr399Ile have been related to susceptibility to gram-negative infections and sepsis and may be involved in the development of atherosclerosis. Identification of TLR4 Asp299Gly and Thr399Ile genotypes can be important for examination of genotype/phenotype relationships as well as for individual risk assessment of patients. Methods: TLR4 Asp299Gly and Thr399Ile genotypes were detected by a single tube polymerase chain reaction (PCR), based on exonuclease degradation of dual labelled allele-specific oligonucleotides. The assay results were compared with conventional restriction fragment length polymorphism (RFLP) analysis. Results: Genotypes of 345 individuals were determined simultaneously in a single PCR assay. Allele frequencies for our population were 6.8% for the TLR4 Asp299Gly polymorphism and 6.4% for the Thr399Ile polymorphism. Validation by RFLP analysis revealed a correct detection of all genotypes. Conclusions: We have developed a novel method for the detection of the TLR4 Asp299Gly and Thr399Ile mutations, permitting rapid genotyping which should be useful for large-scale population studies as well as applicable for routine clinical testing.

Single-nucleotide polymorphism genotyping by melting analysis of dual-labeled probes: examples using factor V Leiden and prothrombin 20210A mutations.

The emergence of fluorescence techniques in 96- or 384-well formats has led to high-throughput single-nucleotide polymorphism (SNP) detection methods. Among these, allele discrimination based on real-time PCR technologies has been developed along two main methodologic approaches. In the first approach, a temperature-dependent fluorescent signal is generated by hybridization of a probe at the end of each PCR cycle. The monitoring of fluorescence emission is followed during (1)(2) or at the end of (3)(4) the PCR process. In the latter case, allele detection is achieved by carrying out of a melting curve analysis. In the second approach, exploiting the classic 5′ nuclease assay (TaqMan®) technology, a temperature-independent fluorescence signal is generated during the PCR process by the exonuclease degradation of a hybridized TaqMan probe (5)(6). Except for one method discussed later (3), all of these techniques require the use of two probes for allele differentiation. An allele differentiation study has stressed the risk of artifacts and subsequent erroneous results when a polymorphism is located within the DNA sequence targeted by TaqMan (7). The same study demonstrated the power of methods relying on determination of melting curves. Although other interesting approaches have been reported recently, such as DASH (8), iFret (9), and dipole calculation (10), some of them are technically difficult or time-consuming. In addition, the SYBR Green technique (11) would represent the simplest method to detect point mutations, but it seems to be limited by its lack of ability to differentiate alleles (12). We present here a new methodologic approach that allows high-throughput genotyping of SNPs. Allele differentiation is achieved by the generation of melting curves, with a single dual-labeled probe, at the end of a classic PCR reaction. The reliability of the method is demonstrated for detection of the factor V Leiden …

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus.

The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5'/3' ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems - exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5'/3' ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases.

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of approximately 100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA.

Synthesis and properties of radiolabeled CPTA–oligonucleotides

AbstractA solid phase technique for the preparation of antisense oligodeoxynucleotides (ODNs) is described featuring 5′‐end conjugated 4‐[(1,4,8,11‐tetraazacyclotet‐radec‐1‐yl)‐methyl]benzoic acid (CPTA). Using Fmoc‐protected CPTA–C6 amidite, CPTA was conjugated to ODNs at the end of an automated DNA synthesis. To illustrate successful conjugations, the CPTA–ODNs were labeled with 99mTc using the stannous‐chloride reduction method. The resulting 99mTc complexes showed differences of stability between CPTA‐conjugated and CPTA‐unconjugated as well as 3′‐protected and 3′‐unprotected ODNs. Propane‐1,3‐diol 3′‐modification enhanced efficiently the stability of 99mTc labeled ODN against exonuclease degradation. Fmoc3CPTA‐C6 amidite turned out to be a versatile ligand for radiometal complexation at the 5′‐end. Copyright © 2002 John Wiley & Sons, Ltd.

Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability, and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases.

Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1',2'-oxetane constrained cytidine, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show approximately 3 degrees C drop per modification in melting temperature (Tm) of their hybrid AON-RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON-RNA duplex. An AON with double C modifications along with 3'-DPPZ (dipyridophenazine) conjugation shows the Tm of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis-Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON-RNA duplexes as well as double C modifications along with 3'-DPPZ have catalytic activities (kcat) close to the native. However, the R Nase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for C modified AON-RNA duplexes. All oxetane modified AON-RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. The C modified AONs (with 3'-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. 18). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the R Nase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.

2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: an analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA.

A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.

PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine

The field of DNA vaccines has grown rapidly, and since most such vaccines involve the inoculation of large circular DNA molecules previously propagated in bacteria, several inconveniences (e.g. the presence of antibiotic resistance genes, impurities from bacterial cultures or inefficient uptake of the large and bulky plasmid DNA molecules to the nucleus) are debated. In this study, we have explored the possibility of using smaller and more flexible PCR-generated linear DNA fragments instead. Phosphorothioate (PTO)-modified primers were used successfully to protect the PCR-generated DNA fragments from exonuclease degradation, and by using a nuclear localization signal-peptide to target the linear DNA to the nucleus the immune response against the encoded antigen was further improved. This approach was tested in cell culture using a sensitive reporter system and in vivo with DNA encoding the amino-terminus of the Puumala hantavirus nucleocapsid protein. Our results indicate that linear DNA fragments have a great potential as a genetic vaccine and phosphorothioate modification in combination with a nuclear localization signal peptide increase the stability and targets the linear DNA molecules to the nucleus resulting in an improved biological response examined both in vitro and in vivo.

Antisense hairpin loop oligonucleotides as inhibitors of expression of multidrug resistance-associated protein 1: their stability in fetal calf serum and human plasma.

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.

Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration

The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3′-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5′-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.

The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation.

The major mRNA degradation pathway involves deadenylation of the target molecule followed by decapping and, finally, 5'-->3' exonuclease digestion of the mRNA body. While yeast factors involved in the decapping and exonuclease degradation steps have been identified, the nature of the factor(s) involved in the deadenylation step remained elusive. Database searches for yeast proteins related to the mammalian deadenylase PARN identified the Pop2 protein (Pop2p) as a potential deadenylase. While Pop2p was previously identified as a factor affecting transcription, we identified a non-canonical RNase D sequence signature in its sequence. Analysis of the fate of a reporter mRNA in a pop2 mutant demonstrates that Pop2p is required for efficient mRNA degradation in vivo. Characterisation of mRNA degradation intermediates accumulating in this mutant supports the involvement of Pop2p in mRNA deadenylation in vivo. Similar phenotypes are observed in yeast strains lacking the Ccr4 protein, which is known to be associated with Pop2p. A recombinant Pop2p fragment encompassing the putative catalytic domain degrades poly(A) in vitro demonstrating that Pop2p is a nuclease. We also demonstrate that poly(A) is a better competitor than poly(G) or poly(C) of the Pop2p nuclease activity. Altogether, our study indicates that Pop2p is a nuclease subunit of the yeast deadenylase and suggests that Pop2p homologues in other species may have similar functions.

Polymer support for exonucleolytic sequencing

Different kinds of particles were investigated for their potential use as supports for exonucleolytic sequence analysis. Composite beads composed of an unreactive polystyrene ‘core’ and a ‘shell’ of functionalized silica nanoparticles were found to best fulfill the various prerequisites. The biotin/streptavidin system was used for attachment of DNA to composite beads of 6 μm diameter. Applying M13 ssDNA in extremely high dilution (∼1 molecule versus 100 beads) with internal fluorescent labels, only a small fraction of beads was found to be associated with fluorescent entities, which likely correspond to a very small number of bound DNA molecules per particle. For better selection and transfer of DNA-containing beads into microstructures for exonuclease degradation the loading experiments were repeated with composite beads of 2.3 μm diameter. In this case a covalent bond was formed between carboxylate-functionalized beads and amino-terminated oligonucleotides, which were detected through external labelling with fluorescent nanoparticles interacting with biotinylated segments of the complementary strand.

Developmental control of telomere lengths and telomerase activity in plants.

Telomere lengths and telomerase activity were studied during the development of a model dioecious plant, Melandrium album (syn Silene latifolia). Telomeric DNA consisted of Arabidopsis-type TTTAGGG tandem repeats. The terminal positions of these repeats were confirmed by both Bal31 exonuclease degradation and in situ hybridization. Analysis of terminal restriction fragments in different tissues and ontogenetic stages showed that telomere lengths are stabilized precisely and do not change during plant growth and development. Telomerase activity tested by using a semiquantitative telomerase repeat amplification protocol correlated with cell proliferation in the tissues analyzed. Highest activity was found in germinating seedlings and root tips, whereas we observed a 100-fold decrease in telomerase activity in leaves and no activity in quiescent seeds. Telomerase also was found in mature pollen grains. Telomerase activity in tissues containing dividing cells and telomere length stability during development suggest their precise control during plant ontogenesis; however, the telomere length regulation mechanism could be unbalanced during in vitro dedifferentiation.