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PubMed1994

The in vitro endonuclease activity of gene product A, the large subunit of the bacteriophage lambda terminase, and its relationship to the endonuclease activity of the holoenzyme.

The reaction requirements and kinetic properties of the in vitro endonuclease activity of the bacteriophage lambda terminase and its large subunit, gene product (gp) A, have been analyzed. Optimal cleavage reaction activity for both proteins requires Mg2+, a pH between 8.5 and 9.0, and is enhanced by ATP or ATP analogs. Under these conditions both terminase and gpA generate aberrant nicks in and around cosN. Optimal nicking specificity of terminase is observed under conditions of 50-100 mM salt, 5 mM spermidine, 1.5 mM ATP, and a pH between 7.0 and 7.5. Specific activity of terminase is greatly reduced under these conditions, and gpA is completely inactive at all protein concentrations tested. Under optimal reaction conditions, gpA endonuclease activity differs from that of the holoenzyme in that it can only be detected at high concentrations, is strongly protein concentration-dependent, and can not be stimulated by the Escherichia coli protein integration host factor. Addition of purified gpNu1 partially, but not completely, minimized these differences, suggesting that the role of gpNu1 in the holoenzyme is to modulate the basal endonuclease of gpA.

Bacteriophage lambda↗

PubMed1997

Site-specific endonuclease AbaI from Azospirillum brasilense UQ 1796 is an isoschizomer of endonuclease BclI.

The site-specific endonuclease AbaI was isolated and purified to functional purity from the soil nitrogen-fixing bacterium Azospirillum brasilense UQ 1796. Purification included successive chromatography on columns with phosphocellulose, heparin-Sepharose, and hydroxyapatite. The purified enzyme recognizes the palindromic DNA sequence 5'-T decreases ATCA-3' and cleaves it as shown by the arrow. The isolated enzyme belongs to class II restriction endonuclease and is an isoschizomer of endonuclease BclI. The enzyme of AbaI is active at 26-56 degrees C. The optimal temperature is 48 degrees C and the optimal buffer is LRB.

Azospirillum brasilense↗

PubMed2001

Interaction of human AP endonuclease 1 with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-patch base excision repair.

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.

Carbon-Oxygen Lyases↗

PubMed1978

DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA.

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.

Animals↗

PubMed1998

Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.

Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.

Acids↗

PubMed2002

AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision repair.

Base loss is common in cellular DNA, resulting from spontaneous degradation and enzymatic removal of damaged bases. Apurinic/apyrimidinic (AP) endonucleases recognize and cleave abasic (AP) sites during base excision repair (BER). APE1 (REF1, HAP1) is the predominant AP endonuclease in mammalian cells. Here we analyzed the influences of APE1 on the human BER pathway. Specifically, APE1 enhanced the enzymatic activity of both flap endonuclease1 (FEN1) and DNA ligase I. FEN1 was stimulated on all tested substrates, regardless of flap length. Interestingly, we have found that APE1 can also inhibit the activities of both enzymes on substrates with a tetrahydrofuran (THF) residue on the 5'-downstream primer of a nick, simulating a reduced abasic site. However once the THF residue was displaced at least a single nucleotide, stimulation of FEN1 activity by APE1 resumes. Stimulation of DNA ligase I required the traditional nicked substrate. Furthermore, APE1 was able to enhance overall product formation in reconstitution of BER steps involving FEN1 cleavage followed by ligation. Overall, APE1 both stimulated downstream components of BER and prevented a futile cleavage and ligation cycle, indicating a far-reaching role in BER.

Base Sequence↗

PubMed2003

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

Active Transport, Cell Nucleus↗

PubMed2001

Covalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease.

The PvuII restriction endonuclease has been converted from its natural homodimeric form into a single polypeptide chain by tandemly linking the two subunits through a short peptide linker. The arrangement of the single-chain PvuII (sc PvuII) is (2-157)-GlySerGlyGly-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit and GlySerGlyGly is the peptide linker. By introducing the corresponding tandem gene into Escherichia coli, PvuII endonuclease activity could be detected in functional in vivo assays. The sc enzyme was expressed at high level as a soluble protein. The purified enzyme was shown to have the molecular mass expected for the designed sc protein. Based on the DNA cleavage patterns obtained with different substrates, the cleavage specificity of the sc PvuII is indistinguishable from that of the wild-type (wt) enzyme. The sc enzyme binds specifically to the cognate DNA site under non-catalytic conditions, in the presence of Ca2+, with the expected 1:1 stoichiometry. Under standard catalytic conditions, the sc enzyme cleaves simultaneously the two DNA strands in a concerted manner. Steady-state kinetic parameters of DNA cleavage by the sc and wt PvuII showed that the sc enzyme is a potent, but somewhat less efficient catalyst; the k(cat)/K(M) values are 1.11 x 10(9) and 3.50 x 10(9) min(-1) M(-1) for the sc and wt enzyme, respectively. The activity decrease is due to the lower turnover number and to the lower substrate affinity. The sc arrangement provides a facile route to obtain asymmetrically modified heterodimeric enzymes.

Amino Acid Sequence↗

PubMed2003

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.

Amino Acid Motifs↗

PubMed2001

Endonuclease IV homolog from Dictyostelium discoideum: sequencing and functional expression in AP endonuclease-deficient Escherichia coli.

We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth(-), nfo(-)).

Amino Acid Sequence↗

PubMed1989

Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but beta-elimination and sometimes beta delta-elimination catalysts.

Bacteriophage-T4 UV endonuclease nicks the C(3')-O-P bond 3' to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5')-O-P bond 5' to the AP site, leaving a 3'-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3')-O-P bond 3' to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.

Catalysis↗

PubMed1990

Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV.

DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments. The yeast Saccharomyces cerevisiae repairs such DNA lesions by using a single major enzyme. We have cloned the yeast structural gene (APN1) encoding this AP endonuclease/3'-repair diesterase by immunological screening of a yeast genomic DNA expression library in lambda gt11. Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts. The DNA and predicted amino acid sequences for the APN1 gene are homologous to those for the nfo gene encoding DNA endonuclease IV of Escherichia coli. This conservation of structure between a eukaryotic enzyme and its prokaryotic counterpart underscores the fundamental nature of their roles in DNA repair.

Amino Acid Sequence↗

PubMed2000

Heat shock protein 70 binds to human apurinic/apyrimidinic endonuclease and stimulates endonuclease activity at abasic sites.

The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10-100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress.

Base Sequence↗

PubMed1983

Does the specific recognition of DNA by the restriction endonuclease EcoRI involve a linear diffusion step? Investigation of the processivity of the EcoRI endonuclease.

The time course of the EcoRI endonuclease catalysed cleavage of three substrates, two plasmid DNAs and one oligonucleotide, each with two EcoRI sites, was measured. The two plasmid DNAs with the EcoRI sites 318 and 96 base pairs apart are cut in a distributive fashion, while the oligonucleotide with the EcoRI sites 8 base pairs apart is cut in a partially processive manner. It is concluded that a linear diffusion of the EcoRI endonuclease on its substrate across long stretches of DNA is not likely to be operative during the recognition process. Microscopic dissociation-reassociation processes, however, increase the probability of the enzyme to attack further sites located in the immediate vicinity of a given site.

Base Composition↗

PubMed1992

Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII.

A study was made of the interaction between restriction endonucleases recognizing CCNGG (SsoII and ScrFI) or CCA/TGG (MvaI and EcoRII) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy] methylguanine (gIG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for gIG) introduced into the recognition site both increase the efficiency of SsoII and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by SsoII occurs at the phosphodiester bond adjacent to the point of modification towards the 5'-end. With the guanine base returned (the substrate with gIG), the correct cleavage position is restored. ScrFI specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the gIG-containing duplex) are resistant to hydrolysis by MvaI and EcoRII. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that SsoII forms a complex with DNA in an 'open' form.

Base Sequence↗

PubMed1999

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.

We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction. The protein was identified as one of the two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning. These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively. The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases. The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II. They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins. The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined. The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein.

Amino Acid Motifs↗

PubMed2004

Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress.

Mitochondrial DNA (mtDNA) is exposed to reactive oxygen species (ROS) produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions, including oxidized pyrimidines, in mtDNA may lead to structural genomic alterations, mitochondrial dysfunction and associated degenerative diseases. In Escherichia coli, oxidative pyrimidines are repaired by endonuclease III (EndoIII) and endonuclease VIII (EndoVIII). To determine whether the overexpression of two bacterial glycosylase/AP lyases which predominantly remove oxidized pyrimidines from DNA, could improve mtDNA repair and cell survival, we constructed vectors containing sequences for the EndoIII and EndoVIII downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) and placed them under the control of the tetracycline (Tet)-response element. Successful integrations of MTS-EndoIII or MTS-EndoVIII into the HeLa Tet-On genome were confirmed by Southern blot. Western blots of mitochondrial extracts from MTS-EndoIII and MTS-EndoVIII clones revealed that the recombinant proteins are targeted into mitochondria and their expressions are doxycycline (Dox) dependent. Enzyme activity assays and mtDNA repair studies showed that the Dox-dependent expressions of MTS-EndoIII and MTS-EndoVIII are functional, and both MTS-EndoIII and MTS-EndoVIII (Dox+) clones were significantly more proficient at repair of oxidative damage in their mtDNA. This enhanced repair led to increased cellular resistance to oxidative stress.

Cell Survival↗

PubMed2003

The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection.

Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and, more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.

Algorithms↗