Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.
...
PMID:The genetic dependence of recombination in recD mutants of Escherichia coli. 306 39

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.
...
PMID:Repair of DNA-containing pyrimidine dimers. 329 78

This paper describes the construction of a DNA molecule containing a topologically stable structure that simulates a replication fork. This preformed DNA molecule is a circular duplex of 7.2 X 10(3) base pairs (M13mp6 DNA) from which arises, at a unique BamHI recognition site, a noncomplementary 5'-phosphoryl-terminated single strand of 237 nucleotides (SV40 DNA). This structure has two experimental attributes. 1) Templates for both leading and lagging strand synthesis exist as stable structures prior to any DNA synthesis. 2) DNA synthesis creates a cleavage site for the restriction endonuclease BamHI. Form I of T7 DNA polymerase, alone, catalyzes limited DNA synthesis at the preformed replication fork whereas Form II, alone, polymerizes less than 5 nucleotides. However, when T7 gene 4 protein is present, Form II of T7 DNA polymerase catalyzes rapid and extensive synthesis via a rolling circle mode. Kinetic analysis of this synthesis reveals that the fork moves at a rate of 300 bases/s at 30 degrees C. We conclude that the T7 gene 4 protein requires a single-stranded DNA binding site from which point it translocates to the replication fork where it functions as a helicase. The phage T4 DNA polymerase catalyzes DNA synthesis at this preformed replication fork in the presence of gene 4 protein, but the amount of DNA synthesized is less that 3% of the amount synthesized by the combination of Form II of T7 DNA polymerase and gene 4 protein. We conclude that T7 DNA polymerase and T7 gene 4 protein interact specifically during DNA synthesis at a replication fork.
...
PMID:A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. 688 16

The adeno-associated virus (AAV) Rep78 protein is required for many aspects of AAV's life cycle including its DNA replication and the regulation of its gene expression. Because of increasing utilization of AAV as a gene therapy vector and its possible use as an anti-cancer/anti-viral agent, the complete characterization of its Rep78 regulatory protein is important. In order to study various functional aspects of Rep78, we have cloned and expressed the Rep78 gene in Escherichia coli using an inducible expression plasmid. The entire Rep78 open reading frame (nt 321 to 2185) was cloned into the LacZ inducible expression vector pMALc2. Upon induction of the Ptac promoter with isopropyl thio-beta-D-galactopyranoside (IPTG), Rep78 is produced as a fusion protein with maltose binding protein (MBP). This recombinant MBP-Rep78 protein displayed all the biochemical activities which are described for the wild type protein including binding to the AAV terminal repeats (TR), endonuclease activity, and helicase activity. Furthermore, for the first time, ATP binding by Rep78 is demonstrated.
...
PMID:Cloning, expression and purification of full length Rep78 of adeno-associated virus as a fusion protein with maltose binding protein in Escherichia coli. 769 27

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.
...
PMID:DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect. 792 64

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.
...
PMID:Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins. 793 14

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.
...
PMID:Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex. 803 57

The bacteriophage lambda terminase is composed of two subunits, gpNu1 and gpA. In vitro, the holoenzyme is a site-specific endonuclease, helicase, ATPase, and can package lambda DNA into proheads. gpA possesses ATPase and helicase activities which are similar to those of the holoenzyme. Both terminase and gpA can hydrolyze a wide range of deoxyribo- and ribonucleoside triphosphates to inorganic phosphate and the corresponding diphosphate. Nucleoside diphosphates are not substrates for either protein. ATPase of both proteins is stimulated by double-stranded DNA. The ATPase of gpA is protein concentration-dependent, while that of terminase is not. Helicase activity of both proteins is not concentration-dependent, and requires a hydrolyzable triphosphate. ATP, dATP, and GTP supported helicase activity, while adenosine 5'-(beta, gamma-methylene)triphosphate, adenosine 5'-3-O-(thio)triphosphate, ADP, CTP, and UTP did not. The kinetic parameters of ATPase and helicase activities were similar for both proteins, but packaging with terminase was optimal only at a significantly higher level of ATP. Packaging was detectable at significant levels with CTP and UTP, but not with GTP. Packaging also differed from ATPase and helicase in the utilization of divalent metal cations and susceptibility to various inhibitors.
...
PMID:The in vitro ATPases of bacteriophage lambda terminase and its large subunit, gene product A. The relationship with their DNA helicase and packaging activities. 817 94

An activity gel assay was developed for the detection of DNA helicases in crude extracts. The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel. The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography. Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands. A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli. Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies.
...
PMID:An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts. 820 62

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.
...
PMID:Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli. 828 83


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---