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

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

Holliday junctions in DNA are generated as a product of homologous recombination events. To test the hypothesis that human p53 may bind to Holliday junctions, synthetic junctions with four approximately 75-base pair (Hol75) or approximately 565-base pair (Hol565) arms were generated. As seen by electron microscopy, under conditions in which 50-61% of the Hol565 DNAs were bound by p53, 80-96% of the p53 was located specifically at the junction with, in the latter case, only 4% of the p53 visualized at the DNA ends or along the arms. Given the large number of potential binding sites, this represents very high specificity for the junctions. Gel retardation assays using the Hol75 DNA confirm these observations, and indicate that the tight junction complexes have a half-life of greater than 4 h. The binding of p53 to three-way junctions is severalfold less than to four-way junctions. Addition of p53 greatly increases the rate of resolution of the Hol75 DNA by T4 endonuclease VII and T7 endonuclease I, two enzymes known to cleave such junctions. This latter finding further confirms the interaction of p53 with Holliday junctions and suggests that p53 binding facilitates their resolution in vivo.
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PMID:Human p53 binds Holliday junctions strongly and facilitates their cleavage. 905 58

DNA branch migration is a fundamental process in genetic recombination. A new model system has been developed for studying branch migration in a small synthetic four-arm junction. A mathematical method for describing branch-point movement by discrete steps in such junctions is also presented. The key to our experimental system is the ability to fix the location of the branch point during the assembly of the junction with a reversible block. The block is provided by a short oligonucleotide that forms triplex DNA adjacent to the initial location branch point at low pH. Raising the pH causes the triplex strand to dissociate, making the branch point free to migrate. Once mobile, the branch point can run off the end of the junction. The time-course for this runoff is consistent with a random walk of the branch point. If it is assumed that one migration step moves the branch point one base-pair, the time-course gives a rate constant for one step of 1.4 second-1 at 37 degrees C in 10 mM MgCl2, 50 mM NaCl. These values are consistent with other measurements of non-enzymatic branch migration. We have also monitored the spread of the branch points directly with T4 endonuclease VII. Using EcoRI restriction endonuclease, we have shown that the binding of this protein to the arms of the junction essentially blocks branch migration through the binding site. In these experiments Ca2+ replaces Mg2+, and the enzyme does not cleave the DNA. In vivo there must be a special process to get branch points to migrate past bound proteins.
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PMID:Triple-helical DNA as a reversible block of the branch point in a partially symmetrical DNA four-arm junction. 926 64

The function of the Lactococcus lactis bacteriophage bIL66 middle time-expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined. Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and is lethal to recA and recBC E. coli mutants, which are both deficient in recombinational repair of chromosomal double-stranded breaks (DSBs). The use of an inducible expression system allowed us to demonstrate that the M-operon-encoded proteins generate a limited number of randomly distributed chromosomal DSBs that are substrates for ExoV-mediated DNA degradation. DSBs were also shown to occur upstream of the replication initiation point of unidirectionally theta-replicating plasmids. The characteristics of the DSBs lead us to propose that the endonucleolytic activity of the M-operon is not specific to DNA sequence, but rather to branched DNA structures. Genetic and physical analysis performed with different derivatives of the M-operon indicated that two orfs (orf2 and orf3) are needed for nucleolytic activity. The orf3 product has amino acid homology with the E. coli RuvC Holliday junction resolvase. By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) and conserved in ORF3 (Glu-67) is essential for the nucleolytic activity of the M-operon gene product(s). We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (M-nuclease), which might be essential for phage multiplication.
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PMID:Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC. 964 49

Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a Kd of binding to Holliday junctions of 0.5 nM. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies.
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PMID:'Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes. 1006 81

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1:ura4+ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4- strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4+ strain. Possible explanations are discussed.
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PMID:The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe. 1095 73

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.
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PMID:SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. 1098 59

The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.
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PMID:Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities. 1100 13

The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal. Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of SULFOLOBUS:, the Holliday junction resolving activity detected in P. furiosus cell extract thus far is only derived from Hjc.
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PMID:Biochemical characterization of the hjc holliday junction resolvase of Pyrococcus furiosus. 1107 44

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.
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PMID:Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide. 1111 9

In homologous recombination in bacteria, the RuvAB Holliday junction-specific helicase catalyzes Holliday junction branch migration, and the RuvC Holliday junction resolvase catalyzes formation of spliced or patched structures. RuvAB and RuvC from the hyperthermophile Thermotoga maritima were expressed in Escherichia coli and purified to homogeneity. An inverted repeat sequence with unique termini was produced by PCR, restriction endonuclease cleavage, and head-to-tail ligation. A second inverted repeat sequence was derived by amplification of a second template containing a three-nucleotide insertion. Reassociation products from a mixture of these two sequences were homoduplex linear molecules and heteroduplex heat-stable Holliday junctions, which acted as substrates for both T. maritima RuvAB and RuvC. The T. maritima RuvAB helicase catalyzed energy-dependent Holliday junction branch migration at 70 degrees C, leading to heteroduplex linear duplex molecules with two three-nucleotide loops. Either ATP or ATP gamma S hydrolysis served as the energy source. T. maritima RuvC resolved Holliday junctions at 70 degrees C. Remarkably, the cleavage site was identical to the preferred cleavage site for E. coli RuvC [(A/T)TT(downward arrow)(G/C)]. The conservation of function and the ease of purification of wild-type and mutant thermophilic proteins argues for the use of T. maritima proteins for additional biochemical and structural studies.
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PMID:The ruv proteins of Thermotoga maritima: branch migration and resolution of Holliday junctions. 1112 78


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