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PubMed1984

Cytoskeletal involvement in cAMP-induced sensitization of chromatin to nuclease digestion in transformed Chinese hamster ovary K1 cells.

The cAMP-induced reverse transformation of CHO-K1 cells, which restores fibroblastic morphology, normal nuclear structure, specific membrane structures and biochemical activities, and cell growth regulation, also restores the sensitivity of nuclear chromatin digestion by DNase I to that resembling the normal fibroblast. All of these aspects of the reverse-transformation reaction require integrity of the cytoskeleton. The nuclease-sensitivity effect is achieved only when the entire cell rather than the isolated nucleus is incubated with cAMP derivatives, indicating linkage between the cytoskeleton and nuclear components. Evidence is presented to show that the DNA sensitization to digestion involves interaction between DNA and other chromatin components and affects different regions of the genome in specific ways. Normal fibroblasts display greater endogenous nuclease activity than the transformed cell. The data are interpreted in terms of a genetic regulatory system extending from the membrane to the nucleus and utilizing the cAMP-induced cytoskeleton.

Animals↗
PubMed1985

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

UvrABC excision nuclease (UvrA, UvrB, and UvrC proteins) of Escherichia coli removes nucleotide mono- and diadducts from DNA in the form of oligonucleotides 12 or 13 bases long. We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence of other proteins implicated in excision repair. Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.

Adenosine Triphosphatases↗
PubMed1993

Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease.

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.

Animals↗
PubMed1993

Identification and characterization of a nuclease activity specific for G4 tetrastranded DNA.

We have identified a nuclease activity that is specific for G4 tetrastranded DNA. This activity, found in a partially purified fraction for a yeast telomere-binding protein, binds to DNA molecules with G4 tetrastranded structure, regardless of their nucleotide sequences, and cleaves the DNA in a neighboring single-stranded region 5' to the G4 structure. Competition with various G4-DNA molecules inhibits the cleavage reaction, suggesting that this nuclease activity is specific for G4 tetrastranded DNA. The existence of this enzymatic activity that reacts with G4 DNAs but not with single-stranded or Watson-Crick duplex DNAs suggests that tetrastranded DNA may have a distinct biological function in vivo.

Base Sequence↗
PubMed1994

Excision close to matrix attachment regions of the entire chicken alpha-globin gene domain by nuclease S1 and characterization of the framing structures.

Nuclease S1-hypersensitive sites in a 40-kb region of the chicken genome including the domain of the alpha-globin genes were mapped. Brief treatment of isolated chicken erythroid cell nuclei with nuclease S1 allowed separation of an approximately 20-kb genomic DNA fragment containing the whole alpha-globin gene cluster. No S1-hypersensitive sites were observed in the internal part of the domain. The upstream S1 site was found in a DNA fragment of 1.7 kb where the origin of replication and several protein binding sites were identified previously. Precise mapping of the positions of S1 cleavage in this fragment and "in vivo" footprinting of DNA-protein interactions in isolated nuclei showed a correspondence with some of these protein binding sites. The possible significance of all these observations is discussed in connection with the replication origin and the nuclear matrix attachment regions in the framing structures.

Animals↗
PubMed1996

Linker histones affect patterns of digestion of supercoiled plasmids by single-strand-specific nucleases.

The effect of histone H1 binding on the cleavage of superhelical plasmids by single-strand-specific nucleases was investigated. Mapping of P1 cleavage sites in pBR322, achieved by EcoRI digestion after the original P1 attack, showed an intriguing phenomenon: preexisting susceptible sites became "protected," whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-strand-specific nuclease, S1. Disappearance of cutting at preexisting sites and appearance of new sites was also observed in a derivative plasmid that contains a 36-bp stretch of alternating d(AT) sequence that is known to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)18-containing plasmid led to protection of all preexisting sites except the d(AT)18 inserts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explained by long-range effects of histone H1 binding on the superhelical torsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific sequences in Pl-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-terminal tails is indicated by the fact that titration with the globular domain of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.

Binding Sites↗
PubMed1998

The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli.

The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3' --> 5' exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (chi). We have prepared a RecB deletion mutant (RecB1-929) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB1-929 protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB1-929 with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB1-929CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD's transformation from a 3' --> 5' exonuclease to a helicase when it meets a chi site.

DNA Helicases↗
PubMed1995

Human DNA repair excision nuclease. Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies.

Human DNA repair excision nuclease removes DNA damage by incising on both sides of the lesion in a precise manner. The activity requires participation of 16-17 polypeptides. Of these, the XPF.ERCC1 complex and XPG were predicted to carry the nuclease active sites based on studies with the recombinant proteins and the yeast homologs of these proteins. Furthermore, recent work with model (undamaged) substrates have led to predictions of the roles of these proteins in incising 5' or 3' to the lesion. We have used damaged DNA substrates and antibodies to XPG and ERCC1 to test these predictions. Our results reveal that anti-XPG antibodies change the site of 3' incision and at high concentration inhibit the 3' incision without significantly affecting the 5' incision, indicating that XPG makes the 3' incision and further that under this condition 5' incision can occur without 3' incision. In contrast, anti-ERCC1 antibodies inhibit both the 3' and 5' incisions. Using a defined system for excision repair we also demonstrate that the 3' incision can occur without the 5' incision, leading us to conclude that under certain conditions the two incisions can occur independently.

Animals↗
PubMed1996

Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide.

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Antiviral Agents↗
PubMed1996

Overproduction, purification, and characterization of the XPC subunit of the human DNA repair excision nuclease.

Xeroderma pigmentosum complementation group C gene (XPC) encodes a protein of 125 kDa which is present in a tight complex with a 58-kDa protein encoded by the human homolog of the yeast RAD23 gene, HHR23B (Masutani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P. J., Bootsma, D., Hoeijmakers, J. H. J., and Hanaoka, F.(1994) EMBO J. 13, 1831-1843). The XPC-HHR23B complex is required for excision of thymine dimers from DNA in a human excision nuclease system reconstituted from purified proteins. In order to understand the role of the XPC-HHR23B complex in excision repair, we have overexpressed each subunit alone and the heterodimer in heterologous systems, purified them, and characterized their biochemical properties. We find that both XPC and the heterodimer bind DNA with high affinity and UV-damaged DNA with slightly higher preference. Surprisingly, we find that the XPC subunit alone is sufficient for reconstitution of the human excision nuclease and that the HHR23B subunit has no detectable effect on the excision activity of the reconstituted system.

Animals↗
PubMed1997

Characterization of reaction intermediates of human excision repair nuclease.

Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA.protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC.HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes.

Base Sequence↗
PubMed1998

Impaired mismatch extension by a herpes simplex DNA polymerase mutant with an editing nuclease defect.

The D368A mutation within the 3'-5'-exonuclease domain of the herpes simplex type 1 DNA polymerase inactivates this nuclease and severely interferes with virus viability. Compared with the wild type enzyme, the D368A mutant exhibits substantially elevated rates of incorrect nucleotide incorporation, as measured in a LacZ reversion assay. This high rate occurs in the presence of high levels of dNTPs, a condition that forces the enzyme to extend mismatched primers. Hence, the mutant fails to correct many misincorporations that are removed in the wild type. In addition, the mutant shows a much reduced ability to replicate DNA templates primed with a 3'-mismatch as compared with wild type. This extension defect also appears more severe than observed for replicases which naturally lack editing nucleases. Based on these findings, we suggest that the inability of the D368A herpes simplex mutant polymerase to replicate beyond a mismatched base pair severely inhibits viral replication.

Amino Acid Sequence↗
PubMed1999

Conserved residues of human XPG protein important for nuclease activity and function in nucleotide excision repair.

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

Amino Acid Sequence↗
PubMed2000

Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates.

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.

Apoptosis↗
PubMed2000

Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures.

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.

Antibodies, Monoclonal↗
PubMed2000

Molecular characterization of a hyperinducible, surface membrane-anchored, class I nuclease of a trypanosomatid parasite.

The 3'-nucleotidase/nuclease (3'-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3'-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3'-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an approximately 1000-fold up-expression of 3'-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3'-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3'-NT/NU-expressed protein possessed both 3'-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3'-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the approximately 100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3'-NT/NU enzyme activity was mediated at the posttranscriptional level.

Adenosine Monophosphate↗
PubMed2000

Significant interference with hepatitis B virus replication by a core-nuclease fusion protein.

Hepatitis B virus (HBV), a small DNA containing virus that replicates via reverse transcription, causes acute and chronic B-type hepatitis in humans. The limited success of current therapies for chronic infection has prompted exploration of alternative strategies. Capsid-targeted viral inactivation is a conceptually powerful approach that exploits virion structural proteins to target a degradative enzyme specifically into viral particles. Its principal feasibility has been demonstrated in retroviral model systems but not yet for a medically relevant virus outside the retrovirus family. Recently, we found that C proximal fusion to the HBV capsid protein of the Ca(2+)-dependent nuclease (SN) from Staphylococcus aureus yields a chimeric protein, coreSN, that in Escherichia coli coassembles with the wild-type capsid protein into particles with internal SN domains. Here we show that, in HBV co-transfected human hepatoma cells, less than 1 coreSN protein per 10 wild-type core protein subunits reduced titers of enveloped DNA containing virions by more than 95%. The antiviral effect depends on both an enzymatically active SN and on the core domain. CoreSN does not block assembly of RNA containing nucleocapsids but interferes with proper synthesis of viral DNA inside the capsid, or leads to rapid DNA degradation. Our data suggest an intracellular nuclease activation that, owing to the characteristics of HBV morphogenesis, is nonetheless highly virus specific. HBV may therefore be particularly vulnerable to the capsid-targeted viral inactivation approach.

Antiviral Agents↗
PubMed2004

The hTERT gene is embedded in a nuclease-resistant chromatin domain.

Normal human cells rarely undergo spontaneous immortalization. Given that ectopic expression of the human telomerase catalytic subunit hTERT leads to cellular immortalization, the endogenous hTERT gene is likely constitutively repressed. Hence, we have examined the chromatin structure of the native hTERT locus and the neighboring loci, CRR9 and Xtrp2, in normal human fibroblasts and a set of immortal lines. Using generalized DNase I sensitivity assays, we revealed that the entire hTERT gene was embedded in a chromatin domain that was as resistant to the nuclease as the well studied beta-globin loci in both telomerase-positive and -negative cells. This condensed domain was at least 100 kb in size and contained the intergenic region 5' to the hTERT gene and the downstream Xtrp2 locus. A transition from the nuclease-sensitive CRR9 locus to the condensed region appeared near the 3'-end of the CRR9 gene. hTERT transcription was associated with the appearance of a major DNase I-hypersensitive site positioned around the hTERT transcription start site and several minor hypersensitive sites. In telomerase-negative cells, the inhibition of histone deacetylases by trichostatin A led to the opening of this chromatin domain, accompanied by transcription from the hTERT gene but not the Xtrp2 gene. In contrast, the inhibition of protein synthesis by cycloheximide induced transcription from both the hTERT and Xtrp2 genes, indicating that histone deacetylases and labile factors coordinate to silence this chromosomal region. Taken together, our data suggest a novel mechanism of hTERT regulation at the chromatin level and have important implications for studying telomerase expression.

Binding Sites↗