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PubMed2006

Metal-binding and nuclease activity of an antimicrobial peptide analogue of the salivary histatin 5.

The salivary antimicrobial peptide histatin 5 is characterized by its cationic nature, structural flexibility, and the presence of two metal-binding sites (the ATCUN motif and a Zn-binding motif). These properties make this peptide a good model for the design of new drugs of low molecular weight. In this work, we have synthesized and studied a new peptide, an analogue of the histatin 5 named ATCUN-C16, which contains both metal-binding centers. The results show that our 20-residue-derived peptide preserves anticandidal activity and exhibits a higher propensity to assume a stable conformation in a hydrophobic environment than do histatin 5 and the C16 peptide that contains the 16 residues of the C-terminal part of histatin 5, although overall our peptide remains a flexible molecule. ACTUN-C16 was found to bind DNA in a gel retardation assay and to have a nuclease activity in the presence of copper and zinc ions and ascorbate. Its nuclease activity can be attributed to the synergistic action of oxidative and hydrolytic activities due to the Cu-ATCUN complex and to the zinc ion coordination, respectively. The results show a new property of this family of salivary peptides and suggest a novel use of this peptide as a small nuclease and biotechnological tool.

Amino Acid Sequence↗

PubMed1996

Role of calf RTH-1 nuclease in removal of 5'-ribonucleotides during Okazaki fragment processing.

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.

Animals↗

PubMed1993

Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets.

"Uniformly" modified phosphodiester or phosphorothioate oligonucleotides incorporating 2'-deoxy-2'-fluoroadenosine, -guanosine, -uridine, and -cytidine, reported herein for the first time, when hybridized with RNA afforded consistent additive enhancement of duplex stability without compromising base-pair specificity. CD spectra of the 2'-deoxy-2'-fluoro-modified oligonucleotides hybridized with RNA indicated that the duplex adopts a fully A-form conformation. The 2'-deoxy-2'-fluoro-modified oligonucleotides in phosphodiester form were not resistant to nucleases; however, the modified phosphorothioate oligonucleotides were highly nuclease resistant and retained exceptional binding affinity to the RNA targets. The stabilizing effects of the 2'-deoxy-2'-fluoro modifications on RNA-DNA duplexes were shown to be superior to those of the 2'-O-methylribo substitutions. RNA hybrid duplexes with uniformly 2'-deoxy-2'-fluoro-modified oligonucleotides did not support HeLa RNase H activity; however, incorporation of the modifications into "chimeric" oligonucleotides has been shown to activate mammalian RNase H. "Uniformly" modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides afforded antisense molecules with (1) high binding affinity and selectivity for the RNA target and (2) stability toward nucleases.

Base Sequence↗

PubMed2003

OsSEND-1: a new RAD2 nuclease family member in higher plants.

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H2O2, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.

Amino Acid Sequence↗

PubMed2000

Rapid genotyping by MALDI-monitored nuclease selection from probe libraries.

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5'-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (> or = 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).

Alleles↗

PubMed1996

The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination.

BACKGROUND: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication. In Escherichia coli, this palindrome-mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease. Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants. RESULTS: We have purified the E. coli SbcCD protein from an overexpressing strain and have shown that it has an ATP-dependent DNA double-strand exonuclease activity. Co-purification of nuclease with protein, antibody inhibition and the absence of activity in extracts lacking the sbcCD genes confirm that the activity is intrinsic to SbcCD. The purified protein also has an ATP-independent single-strand DNA endonuclease activity. CONCLUSIONS: We have shown that sbcCD encodes a nuclease. The purified protein has double-strand DNA exonuclease and single-strand DNA endonuclease activities. We propose that SbcCD cleaves secondary structures formed at replication forks and that the broken forks can be repaired by homologous recombination.

Adenosine Triphosphate↗

PubMed2006

Repair of DNA-polypeptide crosslinks by human excision nuclease.

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome.

Amino Acid Sequence↗

PubMed1974

Dissection of chromosome structure with trypsin and nucleases.

Exhaustive digestion of chromatin with trypsin leads to the cleavage of only 20-30 amino acids from each of histones III (f3), IV (f2a1), IIb2 (f2b), and IIb1 (f2a1), the remainder of these chains being resistant. This resistance is not altered by removing the histones from the DNA with 2 M NaCl, but is dramatically reduced in 6 M urea. Histones III, IV, IIb2, and possibly IIb1 are cleaved at their N-termini. Histones I and V and the nonhistone proteins are the first to be attacked by trypsin and have no detectable trypsin-resistant fragments. The arginine rich histones, III and IV, are then cleaved as a pair, followed by most of IIb2 and IIb1, also as a pair. This data is consistent with a model in which basic N-terminal "arms" extend from a trypsin-resistant histone complex. The structural arrangement of these arms relative to the trypsin-resistant histone complex may define the spatial coordinates of DNA binding sites and, consequently, the folding of the DNA fiber in the chromosome. Accompanying the tryptic digestion of the N-terminals of histones III, IV, IIb2, and possibly IIb1, is an increased sensitivity of chromatin to staphylococcal nuclease. As analyzed by electrophoresis, untrypsinized chromatin is digested into eight discrete limit-digest fragments by nuclease. Trypsinization results in the nuclease digestion of some, but not all, of these DNA bands. Together with the information on the way trypsin cleaves histones in chromatin, the analysis of the resistant DNA suggests that histone N-terminals are associated with some DNA bands and histone C-terminals with other DNA bands. We propose that histones fold the chromosome by crosslinking the DNA corresponding to these bands.

Animals↗

PubMed1984

Two-dimensional S1 nuclease heteroduplex mapping: detection of rearrangements in bacterial genomes.

A method of two-dimensional S1 nuclease heteroduplex mapping was developed to detect gene rearrangements and repeated sequences in total bacterial chromosomes. To detect DNA rearrangements between two variant bacterial strains, total chromosomal DNA preparations from the two strains are digested with four-base-recognizing restriction enzymes, mixed together, denatured, renatured, and separated on first-dimension polyacrylamide slab gels. Gel strips are cut out and soaked in a buffer containing S1 nuclease, which diffuses into the strips and digests the DNA fragments at single-stranded regions. The digested DNA is then electrophoresed in a second dimension perpendicular to the first dimension. DNA heteroduplexes that were digested by the S1 nuclease are resolved as distinct spots below a bright unresolved band of homoduplex. This report describes testing of this method on a model system consisting of two nearly isogeneic strains of Escherichia coli, and the application of this method in detecting DNA rearrangements associated with phase variation in Myxococcus xanthus.

Chromosome Mapping↗

PubMed1985

A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site.

We have devised a method for mapping sites on DNA within the nucleus that are protected against nuclease attack by interaction with bound protein or other factors. This "footprinting" method uses an end-labeled sequence-specific DNA probe, which is annealed to the DNA from nuclear digests under carefully controlled conditions. The annealed complexes are treated with single-strand-specific nuclease, the resulting duplex molecules are electrophoresed on gels, and the gels are autoradiographed. The high sensitivity and resolution of the method have made it possible to obtain a detailed map of DNase I cutting patterns in the 5' flanking sequence of the chicken adult beta (beta A)-globin gene within nuclei from various tissues. In nuclei from adult erythrocytes, this domain is hypersensitive to nucleases. However, we detect within the domain two well-defined regions that are protected against attack, indicating the presence of one or more bound factors. Nuclei from oviduct or 5-day-old embryonic erythrocytes, in which the domain is not hypersensitive, show limited and different patterns of protection.

Animals↗

PubMed1986

Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.

High-affinity nucleoprotein acceptor sites for the avian oviduct progesterone receptor (PR) have been enriched by a combination of nuclease digestion and centrifugation. These enriched binding elements exhibited markedly enhanced PR binding on a per mass DNA basis compared to chromatin (20- to 25-fold) or dehistonized chromatin (4- to 5-fold). Electrophoretic analysis of the nuclease-resistant DNA showed that there is a set of DNA fragments of 100-150 base pairs that are protected from digestion. Excessive digestion resulted in smaller DNA fragments and a loss of PR binding activity. The PR binding was saturable using a crude receptor preparation and displayed a competition with the same receptor preparation that was labeled with nonradioactive progesterone. The enhanced binding was also demonstrable using highly purified receptor preparations that exhibit two classes of binding sites both of which are of high affinity and saturable as assessed by Scatchard analyses. These two high-affinity classes of binding sites are shown to be competed by unlabeled purified PR. The nuclease resistance of these nucleoprotein acceptor sites from chromatin is a property similar to the nuclear matrix binding sites suggesting a relationship between these two classes of nuclear acceptor sites.

Animals↗

PubMed1988

A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs.

We have identified a nucleolytic activity in Escherichia coli infected by bacteriophage T4 that introduces cuts in the ribosome binding site of at least two T4 mRNAs. Cutting takes place specifically in the GGAG sequences that are complementary to the 3' end of 16S rRNA (Shine-Dalgarno sequence). The nature of this nucleolytic cut has been investigated by reverse transcriptase mapping, anti-mRNA mapping, utilization of the vaccinia virus guanylyltransferase, and labeling by polynucleotide kinase. We have compared the sequences of target mRNAs with an mRNA of similar sequence but that is not a substrate for the nuclease. This allowed us to narrow down the possibilities for sequence elements that determine nuclease recognition. We hypothesize that this nuclease plays a physiological role in the inhibition of expression of a class of phage proteins.

Base Sequence↗

PubMed1997

Bioactive and nuclease-resistant L-DNA ligand of vasopressin.

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

Animals↗

PubMed1998

Assembly, subunit composition, and footprint of human DNA repair excision nuclease.

The assembly and composition of human excision nuclease were investigated by electrophoretic mobility shift assay and DNase I footprinting. Individual repair factors or any combination of up to four repair factors failed to form DNA-protein complexes of high specificity and stability. A stable complex of high specificity can be detected only when XPA/RPA, transcription factor IIH, XPC.HHR23B, and XPG and ATP are present in the reaction mixture. The XPF.ERCC1 heterodimer changes the electrophoretic mobility of the DNA-protein complex formed with the other five repair factors, but it does not confer additional specificity. By using proteins with peptide tags or antibodies to the repair factors in electrophoretic mobility shift assays, it was found that XPA, replication protein A, transcription factor IIH, XPG, and XPF.excision repair cross-complementing 1 but not XPC.HHR23B were present in the penultimate and ultimate dual incision complexes. Thus, it appears that XPC.HHR23B is a molecular matchmaker that participates in the assembly of the excision nuclease but is not present in the ultimate dual incision complex. The excision nuclease makes an assymmetric DNase I footprint of approximately 30 bp around the damage and increases the DNase I sensitivity of the DNA on both sides of the footprint.

Base Sequence↗

PubMed1996

Reaction mechanism of human DNA repair excision nuclease.

Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3'-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3'-incision precedes the 5'-incision. Consistent with the kinetic data, uncoupled 5'-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease.

Cholesterol↗

PubMed1997

Identification and characterization of a nuclease specific for the 3' end of the U6 small nuclear RNA.

The U6 small nuclear RNA is post-transcriptionally processed by the addition and removal of nontemplate uridylates (Us) at its 3' end prior to incorporation into the U4 x U6 small nuclear ribonucleoprotein complex. An enzyme responsible for removing Us from the U6 3' end has not been previously identified. Here we biochemically isolate and characterize an exonuclease activity from HeLa cells that removes template and nontemplate 3'-nucleotides specifically from U6 RNA. We also report the isolation of an inhibitor of this U6 nuclease. U6 nuclease rapidly removes the four terminal 3' Us found in the human U6 coding sequence in a magnesium-dependent manner. Mutagenesis studies on the U6 RNA define regions essential for processing. U6 nuclease recognizes specific sequences on both strands near the base of the major intramolecular stem loop of the U6 RNA. The preprocessed 3' Us form part of the base of the stem loop, but neither specific sequences nor secondary structure at the four terminal nucleotides are required to achieve processing.

Base Sequence↗

PubMed1999

Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease). Oligomerization and direct interaction with histone H1.

DNA fragmentation factor (DFF) is a heterodimeric protein composed of 45-kDa (DFF45) and 40-kDa (DFF40) subunits, a protein that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals. DFF45 is a specific molecular chaperone and an inhibitor for the nuclease activity of DFF40. Previous studies have shown that upon cleavage of DFF45 by caspase-3, the nuclease activity of DFF40 is relieved of inhibition. Here we further investigate the mechanism of DFF40 activation. We demonstrate that DFF45 can also be cleaved and inactivated by caspase-7 but not by caspase-6 and caspase-8. The cleaved DFF45 fragments dissociate from DFF40, allowing DFF40 to oligomerize to form a large functional complex that cleaves DNA by introducing double strand breaks. Histone H1 directly interacts with DFF, confers DNA binding ability to DFF, and stimulates the nuclease activity of DFF40 by increasing its Kcat and decreasing its Km.

Apoptosis↗

PubMed1999

Order of assembly of human DNA repair excision nuclease.

Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.

DNA Damage↗