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PubMed2003

Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability.

Previous studies have shown that expansion-prone repeats form structures that inhibit human flap endonuclease (FEN-1). We report here that faulty processing by FEN-1 initiates repeat instability in mammalian cells. Disease-length CAG tracts in Huntington's disease mice heterozygous for FEN-1 display a tendency toward expansions over contractions during intergenerational inheritance compared to those in homozygous wild-type mice. Further, with regard to human cells expressing a nuclease-defective FEN-1, we provide direct evidence that an unprocessed FEN-1 substrate is a precursor to instability. In cells with no endogenous defects in DNA repair, exogenous nuclease-defective FEN-1 causes repeat instability and aberrant DNA repair. Inefficient flap processing blocks the formation of Rad51/BRCA1 complexes but invokes repair by other pathways.

Animals↗
PubMed2004

Positive and negative regulation of poly(A) nuclease.

PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

Amino Acid Sequence↗
PubMed1987

Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation.

We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

Animals↗
PubMed1987

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.

Animals↗
PubMed1988

Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression.

X8/6T2, a hamster-human hybrid cell line which contains an inactive human X chromosome, was treated with 5-azacytidine and selected for derepression of hypoxanthine-guanine phosphoribosyltransferase. Clones were examined for coreactivation of the phosphoglycerate kinase gene (Pgk). Of 68 of these hybrids, approximately 20% expressed measurable human phosphoglycerate kinase (PGK) activity. A 600-base-pair region of the Pgk 5' CpG cluster was examined for the methylation status of eight CCGG sites (site 1 being 5'-most) in a number of PGK-negative and PGK-positive cell lines. The inactive X chromosome is normally methylated at all eight sites, and this was also true for the majority of X8/6T2 cells. However, several PGK-negative hybrids were demethylated in the site 3 to site 6 region. PGK activity correlated with demethylation at both sites 6 and 7. The data for PGK-positive and -negative hybrids indicate that demethylation at or near site 7 was necessary for reactivation of Pgk. Chromatin sensitivity to MspI digestion in the nuclei of male lymphoblastoid cells and several PGK-positive and PGK-negative hybrids was examined. PGK-positive cell lines were hypersensitive to digestion, while PGK-negative hybrids were resistant. Cleavage at sites 6 and 7 was observed in all PGK-positive cell lines at each MspI concentration examined. Sites 7 and 8 were less accessible to digestion than site 6. Cleavage in the site 2 to site 5 region was observable at the lowest MspI concentration. In most PGK-positive hybrids, a nonspecific endogenous nuclease detected the presence of a hypersensitive region spanning at least 450 base pairs, bounded at the 3' end near HpaII site 6. Nuclease hypersensitivity appears to be related to promoter activity, because sites 7 and 8 are in transcribed regions of the gene. These data indicate that specific sites within the CpG cluster have a dominant controlling influence over the Pgk promoter conformation and the transcriptional activation of Pgk.

Animals↗
PubMed1988

The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity.

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

DNA Replication↗
PubMed1977

A thermal denaturation study of chromatin and nuclease-produced chromatin fragments.

Calf thymus chromatin and nuclease-produced chromatin fragments have been examined by thermal denaturation measurements. Native chromatin gave a series of distinct melting transitions at 64, 73,79, and 85 degrees C in 0.25 mM EDTA pH8. Treatments such as dialysis, mechanical shearing, or sulfhydryl oxidation of histone H3 carried out on native chromatin significantly altered the derivative melting profiles by blurring the distinct transitions and shifting the highest melting transition to a lower temperature. Derivative melting profiles for electrophoretically purified chromatin fragments, monomer through hexamer, all resembled that obtained from dialyzed chromatin. These results suggest that higher order structures exist in chromatin that are easily disrupted. Since the products of micrococcal nuclease (EC3.1.4.7) digestion of the altered chromatins did not exhibit any major electrophoretic differences from those obtained from nuclei, than most likely the primary arrangements of histones along the DNA are the main determinant for cleavage sites.

Animals↗
PubMed1978

Conformational studies on wheat embryo 5S RNA using nuclease S1 as a probe.

Ribosomal 5S 32 P-labelled RNA was isolated from imbibing wheat embryos and digested with nuclease S1, a single-strand specific nuclease. The products of enzymic digestion were separated by polyacrylamide gel electrophoresis and identified by fingerprint analysis of their RNase T1 digestion products. The results indicate that the most sensitive portion of the molecule, and hence, the region containing the least helical structure, is close to the 5'-terminus. Similarly, the most resistant portion of the molecule is close to, but does not include, the 3'-terminus. These findings are discussed in relation to proposed models for the secondary structure of 5S RNA.

Base Sequence↗
PubMed1994

Role of nucleases in apoptosis.

The last decade has seen the rapid development of research investigating the mechanisms of apoptosis in a variety of experimental systems. Among the multitude of changes observed in apoptotic cells, chromatin cleavage is considered a biochemical hallmark of apoptosis. Chromatin fragmentation is an enzymatic process which depends on the activity of endogenous nuclease(s) and the susceptibility of chromatin to endonuclease activity. The characteristics of some nucleases of potential importance in apoptosis and their possible role in the regulation of this process are discussed in this paper.

Animals↗
PubMed1975

A 3'-deoxymononucleotide-producing nuclease from the marine sponge Verongia aerophoba. I. Purification.

A four-step procedure for purification of a nuclease from the keratinous sponge Verongia aerophoba is described. The extracted material is lyophilized, acidified, and subjected to chromatography on Sephadex, hydroxyapatite, and phosphocellulose. The nuclease is purified about 1,000-fold from the crude extract and approximately 1,600-fold from concomitant acid RNase. Phosphodiesterase is lost after chromatography on Sephadex. The purified enzyme solution contains one single activity as determined by polyacrylamide gel electrophoresis.

Acid Phosphatase↗
PubMed2005

Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site.

BACKGROUND: Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636. RESULTS: Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family. CONCLUSION: After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.

Amino Acid Sequence↗
PubMed2003

Tudor and nuclease-like domains containing protein p100 function as coactivators for signal transducer and activator of transcription 5.

Signal transducer and activator of transcription 5 (Stat5) plays a critical role in prolactin (PRL)-induced transcription of several milk protein genes. Stat5-mediated gene regulation is modulated by cooperation of Stat5 with cell type- and promoter-specific transcription factors as well as by interaction with transcriptional coregulators. Recently, the expression of a tudor and staphylococcal nuclease-like domains containing protein p100 was found to be increased in mammary epithelial cells during lactation in response to lactogenic hormones. p100 was initially identified as a transcriptional coactivator of the Epstein-Barr virus nuclear antigen 2. In this study we investigated the potential role of p100 in PRL-induced Stat5-mediated transcriptional activation. PRL stimulation increased the p100 protein levels in HC11 mouse mammary epithelial cells. p100 did not affect the early activation events of Stat5, but p100 enhanced the Stat5-dependent transcriptional activation in HC11 cells. p100 associated with Stat5 both in vivo and in vitro, and the interaction was mediated by both the tudor and staphylococcal nuclease-like domains of p100. Together these results suggest that p100 functions as a transcriptional coactivator for Stat5-dependent gene regulation and the existence of a positive regulatory loop in PRL-induced transcription, in which PRL stabilizes p100 protein, which in turn can cooperate with Stat5 in transcriptional activation.

Animals↗
PubMed1999

Molecular genetic analysis of the APEX nuclease gene in amyotrophic lateral sclerosis.

We analyzed genomic DNA from ALS patients for mutations in the apurinic/apyrimidinic endonuclease (APEX nuclease) gene. We identified three rare polymorphisms in the untranslated region of the gene and one common two-allele polymorphism (D148E). The allelic frequency D148E was significantly different in sporadic ALS patients compared with controls. A conserved amino acid change and a 4-base pair deletion were also identified in sporadic ALS patients. These data suggest that APEX nuclease may contribute to the etiology of ALS.

Adult↗
PubMed2003

Hiding at the ends of yeast chromosomes: telomeres, nucleases and checkpoint pathways.

Telomeres stabilise DNA at the ends of chromosomes, preventing chromosome fusion and genetic instability. Telomeres differ from double strand breaks in that they activate neither DNA repair nor DNA damage checkpoint pathways. Paradoxically DNA repair and checkpoint genes play critical roles in telomere stability. Recent work has provided insights into the roles of DNA repair and DNA damage checkpoint pathways in the physiological maintenance of telomeres and in cellular responses when telomeres become uncapped. In budding yeast the Mre11p nuclease, along with other unidentified nucleases, plays critical roles in physiological telomere maintenance. However, when telomeres are uncapped, the 5'-to-3' exonuclease, Exo1p, plays a critical role in generating single-stranded DNA and activating checkpoint pathways. Intriguingly Exo1p does not play an important role in normal telomere maintenance. Although checkpoint pathways are not normally activated by telomeres, at least four different types of telomere defect activate checkpoint pathways. Interestingly, each of these telomere defects depends on a different subset of checkpoint proteins to induce cell cycle arrest. A model for how a spectrum of telomeric states might interact with telomerase and checkpoint pathways is proposed.

Cell Cycle↗
PubMed2003

Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions.

During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP). Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches.

Journal Article↗
PubMed1987

32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A 32P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10(7) to 10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are 32P-labeled at 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P1. The latter observation has been utilized to enhance the 32P-assay's sensitivity to 1 adduct in 10(10) nucleotides for a 10-micrograms DNA sample by postincubation of DNA digests with nuclease P1 before 32P-labeling. The enzyme dephosphorylates the normal nucleotides but not most aromatic and bulky nonaromatic adducts, so that only the latter serve as substrates for the kinase-catalyzed labeling reaction. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and in humans.

Animals↗
PubMed2006

Development of a 5'-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples.

BACKGROUND: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. METHODS: We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. RESULTS: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. CONCLUSIONS: Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

Animals↗
PubMedDate not supplied

A nonspecific, single-stranded nuclease activity with characteristics of a topoisomerase found in a major grass pollen allergen: possible biological significance.

The major allergen from timothy grass pollen, Phlp5b (Phleum pratense), was shown to exhibit ribonuclease activity. It turned out that the C-terminal portion of this molecule was the biologically active domain. Here evidence is presented that the allergen is a single-stranded, sugar-nonspecific nuclease with topoisomerase activity. An isomerase-specific active site was identified, and a non-active mutant was constructed by site directed mutagenesis, and showed no nucleolytic activity. In contrast to the wild type (WT), the mutant did not dimerize. Although the binding capacity of IgE antibodies toward the mutant was reduced as compared to the WT, the allergenic activity was retained. We conclude that the allergen Phlp5b is a single-stranded nuclease with an unusual topoisomerase-like activity. This biological activity is not by itself connected to the allergenicity of the molecule. Whether the enzymatic activity is responsible for the induction of the allergic sensitization and inflammation remains an open question.

Allergens↗