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At least 631 records · Page 35Linked to original sources
PubMed2004

The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2.

Werner syndrome (WS) is characterized by features of premature aging and is caused by loss of the RecQ helicase protein WRN. WS fibroblasts display defects associated with telomere dysfunction, including accelerated telomere erosion and premature senescence. In yeast, RecQ helicases act in an alternative pathway for telomere lengthening (ALT) via homologous recombination. We found that WRN associates with telomeres when dissociation of telomeric D loops is likely during replication and recombination. In human ALT cells, WRN associates directly with telomeric DNA. The majority of TRF1/PCNA colocalizing foci contained WRN in live S phase ALT cells but not in telomerase-positive HeLa cells. Biochemically, the WRN helicase and 3' to 5' exonuclease act simultaneously and cooperate to release the 3' invading tail from a telomeric D loop in vitro. The telomere binding proteins TRF1 and TRF2 limit digestion by WRN. We propose roles for WRN in dissociating telomeric structures in telomerase-deficient cells.

Cell Line, Tumor↗
PubMed2006

Evidence for preferential Ig gene usage and differential TdT and exonuclease activities in human naïve and memory B cells.

Memory B cells and the antibodies they encode are important for protective immunity against infectious pathogens. Characterization of naïve and memory B cell antibody repertoires will elucidate the molecular basis for the generation of antibody diversity in human B cells and the optimization of antibody structures that bind microbial antigens. In this study we aimed to investigate the influence of antigenic selection on the antibody genes of the two CD27+ memory B cell subsets, comparing them with the naïve repertoire in CD27- cells. We analyzed and compared the Ig heavy chain gene transcripts in three recently defined circulating naïve and memory B cell subsets (CD19+IgD+CD27- [naïve], CD19+IgD+CD27+ [un-class-switched memory] or CD19+IgD- CD27+ [class-switched memory]) at the single cell level. We found similar biased patterns of variable, diversity and joining heavy chain gene usages in all three groups of cells. CD19+IgD+CD27+ memory B cells harbored as diverse an antibody gene repertoire as CD19+IgD-CD27+ memory B cells. Interestingly, CD19+IgD+CD27+ memory B cells possessed a lower frequency of somatic mutations, a higher incidence of exonuclease activity at the 3' end of D regions, and a lower frequency of N and P nucleotide additions at both VH-D and D-JH junctions of CDR3 regions compared to CD19+IgD-CD27+ memory B cells. These data suggest distinct functional mechanisms underlying selection of this unique subset of un-class-switched memory B cells.

Adult↗
PubMed2005

The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit.

The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The similarity of B-subunit sequences implies a common fold, but since the key catalytic and metal binding residues of the phosphoesterase domain are disrupted in the eukaryotic B-subunits, their common function has not been identified. To study the structure and activities of B-subunits in more detail, we expressed 13 different recombinant B-subunits in Escherichia coli. We found that the solubility of a protein could be predicted from the calculated GRAVY score. These scores were useful for the selection of proteins for successful expression. We optimized the expression and purification of Methanocaldococcus (Methanococcus) jannaschii DP1 of DNA polymerase D (MjaDP1) and show that the protein co-purifies with a thermostable nuclease activity. Truncation of the protein indicates that the N-terminus (aa 1-134) is not needed for catalysis. The C-terminal part of the protein containing both the calcineurin-like phosphoesterase domain and the OB-fold is sufficient for the nuclease activity.

Archaea↗
PubMed2003

Stimulation of a mitochondrial endo-exonuclease from Podospora anserina by PCNA.

In Podospora anserina we have described the purification of an endo-exonuclease (Biochim. Biophys. Acta 1574 (1) (2002) 72). Given the description of several nucleases addressed to the mitochondria and known to interact with the PCNA, we sought a possible effect of PCNA on the mt nuclease. A significant stimulation of the nuclease activity with PCNA was observed with double-stranded and flap structure DNA. Immuno-Western blotting experiments realized with monoclonal antibodies raised against the PCNA specifically revealed the presence of a single band of 30 kDa in the mitochondria from the filamentous fungus and yeast. A potential PCNA binding motif was found in the sequences of several mt nucleases and mt proteins involved in the maintenance of the mt DNA with respect to the consensus described by Warbrick. The hypothetical role of the PCNA as a potential regulator of the repair/recombination processes in the maintenance of the mt genome is discussed.

Amino Acid Sequence↗
PubMed2003

Identification of EloA-BP1, a novel Elongin A binding protein with an exonuclease homology domain.

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit, and two positive regulatory B and C subunits. Although the NH(2)-terminal approximately 120 amino acid region of Elongin A is dispensable for its transcriptional activity in vitro, it shares significant sequence similarity with the NH(2)-terminus of other class of transcription factors SII and CRSP70, suggesting that the NH(2)-terminus mediates interactions important for the regulation of transcription in vivo. To identify proteins that can bind to these conserved sequences, a human B cell cDNA library was screened using the NH(2)-terminus of Elongin A as bait in a yeast two-hybrid system. Here, we report on the cloning and characterization of a novel human exonuclease domain-containing protein, Elongin A-binding protein 1 (EloA-BP1). EloA-BP1 is composed of 1221 amino acids and its mRNA is ubiquitously expressed. Double immunofluorescence labeling in COS7 cells suggested that EloA-BP1 and Elongin A are colocalized to the cell nucleus. By using an in vitro binding assay, we show that EloA-BP1 is capable of binding not only the NH(2)-terminal approximately 120 amino acid region of Elongin A, but also that of SII. Although the purified EloA-BP1 had no detectable effect on the rate of transcription elongation in vitro, it may play some role in the regulation of elongation in vivo.

Amino Acid Sequence↗
PubMed1984

A 3' to 5' exonuclease activity is associated with phage 029 DNA polymerase.

Bacteriophage 029 produces its own DNA polymerase which is encoded by gene 2 [Watabe, K. and Ito, J. (1983) Nucleic Acid Res. 11, 8333]. This 029 DNA polymerase has been purified by phospho-cellulose, DEAE-cellulose, double-stranded DNA cellulose chromatography and glycerol gradient centrifugation. An exonuclease activity associated with the DNA polymerase was found through all the steps of the purification. This nuclease preferably degrades single-stranded DNA from the 3' to the 5' terminus direction, suggesting that the enzyme plays a role for proofreading during DNA replication. While DNA polymerase activity isolated from cells infected with temperature sensitive mutant of gene 2 is thermolabile, the nuclease activity is not significantly reduced at the restrictive temperature.

Bacillus subtilis↗
PubMed1996

Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins.

Bacteriophage T4 RNase H is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms resolution in the presence of Mg2+ but in the absence of nucleic acids. The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site. This structure suggests the way in which the widely separated conserved regions in the larger nucleotide excision repair proteins, such as human XPG, could assemble into a structure like that of the smaller replication nucleases.

Bacteriophage T4↗
PubMed1998

Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.

Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA) and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat expansions as seen in some cancers and genetic diseases.

Amino Acid Sequence↗
PubMed2000

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue improves the 3'-exonuclease stability of 2'-5'-oligoadenylate-antisense conjugates.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue at the 3'-terminus of DNA or 2-5A-DNA sequences resulted in a significantly enhanced 3'-exonuclease resistance while the affinity for complementary RNA was only slightly decreased. Furthermore, the binding to and activation of human RNase L by thus modified 2-5A-DNA conjugates was not altered as compared to the parent unmodified 2-5A-DNAs.

Adenine Nucleotides↗
PubMed2001

Determination of photomodified oligodeoxynucleotides by exonuclease digestion, matrix-assisted laser desorption/ionization and post-source decay mass spectrometry.

A fast method to detect and sequence photomodified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is reported. Upon treatment of modified ODNs with both phosphodiesterase I and phosphodiesterase II, the digestion stops at the sites of photomodification. Post-source decay (PSD) of MALDI-produced ions from two enzymatic digestion end products distinguishes isomers such as 5'-d(T[cis-syn]TAAGC) and 5'-d(CGAAT[cis-syn]T), which have symmetrical or identical compositions at the 3' and 5' ends, respectively. Studies have also been done to follow the kinetics for enzyme degradation of photomodified ODNs. The calculated rate constants from a mathematical treatment of the time-dependent MALDI data clearly show that the enzymatic digestion rate slows as the enzyme approaches the modified site.

Exonucleases↗
PubMed2005

Loss of DNA minor groove interactions by exonuclease-deficient Klenow polymerase inhibits O6-methylguanine and abasic site translesion synthesis.

The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks template strand interactions. O(6)-Methylguanine (m6G) and abasic site TLS was examined in three stages: dNTP insertion opposite the lesion, extension from a terminal lesion-containing base pair, and the dissociation equilibrium of the polymerase from the lesion-containing template. Less than 5% TLS was observed at the insertion step for either variant on the lesion-containing templates. While extensive TLS was observed for WT polymerase on the m6G template, only incorporation opposite the lesion was observed for the R668A variant. Loss of the template strand interaction, Q849A, resulted in the inability to insert dNTPs opposite either the m6G or abasic lesion. For both variants, extension of purine-containing m6G primer-templates was increased relative to WT polymerase. We observed similar extension efficiencies for all variants, relative to WT, using abasic template-primers. Polymerase dissociation/reassociation was studied through the use of a competitor primer/template complex. Dissociation for WT polymerase increased 2-fold and 3-fold, respectively, for m6G and abasic lesion-containing templates, relative to the natural template. Variants lacking DNA minor groove interactions displayed increased dissociation from DNA templates, relative to WT polymerase, but do not display an increased level of lesion-induced polymerase dissociation. Our results indicate that the primer and template strand interactions of the Klenow polymerase with the DNA minor groove are critical for maintaining the DNA-polymerase complex during translesion synthesis.

Amino Acid Substitution↗
PubMed1996

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

Exonucleases↗
PubMed1997

Synthesis and properties of second-generation 2-5A-antisense chimeras with enhanced resistance to exonucleases.

In order to stabilize 2-5A-antisense chimeras to exonucleases, we have synthesized chimeric oligonucleotides in which the last phosphodiester bond at the 3'-terminus of the antisense domain was inverted from the usual 3',5'-linkage to a 3',3'-linkage. The preparation of such analogues was accomplished through standard phosphoramidite chemistry with the use of a controlled pore glass solid support with a nucleoside attached through its 5'-hydroxyl, thereby permitting elongation at the 3'-hydroxyl. The structures of such terminally inverted linkage chimeras of the general formula pA4-[pBu]2-(pdNn3'-3'dN) were corroborated by a combination of snake venom phosphodiesterase digestion in the presence or absence of bacterial alkaline phosphatase. Most characteristically, the presence of the 3'-terminal-inverted phosphodiester linkage produced an unnatural dinucleotide of general composition dN3'p3'dM. These structures could be confirmed by independent synthesis and fast atom bombardment mass spectroscopy (FAB). 2-5A-Antisense chimeras of this structural class, pA4-[pBu]2-(pdNn'3-3'dN), were 5-6-fold more stable than their unmodified congeners, pA4-[pBu]2-(pdN)n, to degradation by a representative phosphodiesterase from snake venom. In 10% human serum, the new 2-5A-antisense chimeras, pA4-[pBu]2-(pdNn3'-3'dN), possessed a half-life that was 28-fold longer than that of the unmodified chimeras. These results provide entry to a second generation of 2-5A-antisense chimeras.

Adenine Nucleotides↗
PubMed1997

Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization.

In situ hybridization experiments are stringently dependent on the quality of the probes, which should be single-stranded when efficient comparison of signals obtained with antisense and control sense probes are needed. In this report, we describe an optimized synthesis of radioactive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step. The sequence region selected as probe is amplified by polymerase chain reaction in the presence of radiolabelled nucleotides. The sense and antisense probes are then yielded by the action of the lambda bacteriophage exonuclease, which can specifically eliminate one out of the two strands of the amplified fragments. In this way, sense and antisense probes with identical length and specific activity can be generated by selecting the primer to be phosphorylated. We have verified the efficiency of our probes for in situ hybridization of the clusterin transcripts within the peripheral olfactory system, after surgical lesion of its synaptic target.

Animals↗
PubMed2000

5'-->3' exonuclease-based real-time PCR assays for detecting the t(14;18)(q32;21): a survey of 162 malignant lymphomas and reactive specimens.

We describe our experience using two real-time polymerase chain reaction (PCR) assays for detecting the t(14;18)(q32;q21) in a large series of non-Hodgkin's lymphomas (NHLs). These assays utilize the 5'-->3' exonuclease activity of Taq polymerase, which cleaves a probe labeled with a fluorescent reporter dye at its 5' end and a quencher dye at its 3' end during the extension phase of PCR. In a previous study, Luthra and colleagues developed these real-time PCR assays for detecting the t(14;18) involving the major and minor breakpoint cluster regions of the bcl-2 gene and assessed a small number of NHLs. In this larger study, we analyzed 135 NHLs, 6 Hodgkin's disease, 10 reactive biopsy specimens, and 11 peripheral blood specimens. The NHL group included 46 of 70 (65.7%) follicular NHLs, 1 of 2 (50%) diffuse small cleaved cell NHLs, and 13 of 24 (54.2%) diffuse large B-cell NHLs with the t(14;18) detected by conventional PCR methods. There was excellent agreement between the real-time and conventional PCR assays with overall concordance in 160 of 162 (98.8%) specimens. For the NHLs, concordance was found in 134 of 135 (99.3%) specimens. Disagreement was observed in one case of follicular NHL in which the real-time PCR assay detected bcl-2 minor breakpoint cluster region/JH DNA fusion sequences and the conventional method was negative. The overall concordance for 10 benign biopsy specimens and 11 normal peripheral blood samples was 20 of 21 (95.2%). One lymph node biopsy specimen that showed reactive follicular hyperplasia was positive for the bcl-2 minor breakpoint cluster region/JH DNA fusion sequences detected by the real-time PCR assay but was negative by conventional PCR methods. This patient had no clinical evidence of NHL. We conclude that real-time PCR assays for detecting the t(14;18) are sensitive, specific, and more convenient than conventional PCR methods.

Chromosomes, Human, Pair 14↗
PubMed2004

TRF2 recruits the Werner syndrome (WRN) exonuclease for processing of telomeric DNA.

The cancer-prone and premature aging disease Werner syndrome is due to loss of WRN gene function. Cells lacking WRN demonstrate genomic instability, including telomeric abnormalities and undergo premature senescence, suggesting defects in telomere metabolism. This notion is strongly supported by our finding of physical and functional interactions between WRN and TRF2, a telomeric repeat binding factor essential for proper telomeric structure. TRF2 binds to DNA substrates containing telomeric repeats and facilitates their degradation specifically by WRN exonuclease activity. WRN and TRF2 also interact directly in the absence of DNA. These results suggest that TRF2 recruits WRN for accurate processing of telomeric structures in vivo. Thus, our findings link problems in telomere maintenance to both carcinogenesis and specific features of aging.

Base Sequence↗
PubMed2004

The exonuclease ISG20 is directly induced by synthetic dsRNA via NF-kappaB and IRF1 activation.

Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus-host interactions. Recently, we demonstrated that the IFN-induced 3' --> 5' exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-kappaB factors p50 and p65 bind and activate the kappaB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-kappaB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.

Binding Sites↗
PubMed1984

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.

Binding Sites↗