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)

The HO endonuclease was used to introduce a site-specific double-strand break (DSB) in an interval designed to monitor mitotic recombination. The interval included the trp1 and his3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Mitotic recombination was monitored in a diploid carrying heteroalleles of trp1 and his3. The normal recognition sites for the HO endonuclease were mutated at the MAT alleles and a synthetic recognition site for HO endonuclease was placed between trp1 and his3 on one of the chromosomes. HO-induced cleavage resulted in efficient recombination in this interval. Most of the data can be explained by double-strand gap repair in which the cut chromosome acts as the recipient. However, analysis of some of the recombinants indicates that regions of heteroduplex were generated flanking the site of the cut, and that some recombinants were the result of the cut chromosome acting as the genetic donor.
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PMID:Recombination initiated by double-strand breaks. 846 28

Cells of the yeast Saccharomyces cerevisiae are delayed in the G2 phase of the cell cycle following chromosomal DNA damage. This arrest is RAD9-dependent and suggests a signaling mechanism(s) between chromosomal lesions and cell cycling. We examined the global nature of growth inhibition caused by an HO endonuclease-induced double-strand break (DSB) at a 45-bp YZ sequence (from MAT YZ) in a non-yeast region of a dispensable single-copy plasmid. The presence of an unrepaired DSB results in cellular death even though the plasmid is dispensable. Loss of cell viability is partially dependent on the RAD9 gene product. Following induction of the DSB, 40% of RAD+ and 49% of rad9 delta cells [including both unbudded (G1) and budded (S plus G2) cells] did not progress further in the cell cycle. The remaining RAD+ cells progressed to form microcolonies (< 30 cells) containing aberrantly shaped inviable cells. For the rad9 delta mutant, the majority of the remaining cells produced viable colonies accounting for the greater survival of the rad9 delta strain. Based on the profound effects of a single nonchromosomal DNA lesion, this system provides a convenient means for studying the signaling effects of a DNA lesion, as well as for designing strategies for modulating cell proliferation.
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PMID:Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. 851 8

Cell type in the yeast Saccharomyces cerevisiae is determined by information present at the MAT locus. Cells can switch mating types when cell-type information located at a silent locus, HML or HMR, is transposed to the MAT locus. The HML and HMR loci are kept silent through the action of a number of proteins, one of which is the DNA-binding protein, ABF1. We have identified a binding site for ABF1 within the Ya region of MATa and HMRa. In order to examine the function of this ABF1-binding site, we have constructed strains that lack the site in the MATa or HMRa loci. Consistent with the idea that ABF1 plays a redundant role in silencing, it was found that a triple deletion of the ABF1-binding sites at HMRE, Ya and I did not permit the expression of HMRa. We have also shown that chromosomal deletion of the binding site at MATYa had no effect on the level of cutting by the HO endonuclease nor on the amount of mating-type switching observed. Similarly, chromosomal deletion of all three ABF1-binding sites at HMRa had no effect on the directionality of mating-type switching.
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PMID:Functional analysis of the ABF1-binding sites within the Ya regions of the MATa and HMRa loci of Saccharomyces cerevisiae. 853 7

HO-endonuclease initiates a mating-type switch in the yeast S. cerevisiae by making a double-strand cleavage in the DNA of the mating-type gene, MAT. Heterothallic strains of yeast have a stable mating type and contain a recessive ho allele. Here we report the sequence of the ho allele; ho has four point mutations all of which encode for substitute amino acids. The fourth mutation is a leucine to histidine substitution within a presumptive zinc finger. Chimeric HO/ho genes were constructed in vivo by converting different parts of the sequence of the genomic ho allele to the HO sequence by gene conversion. HO activity was assessed by three bioassays: a mating-type switch, extinction of expression of an a-specific reporter gene, and the appearance of Canr Ade- papillae resulting from excision of an engineered Ty element containing the HO-endonuclease target site and a SUP4 degrees gene. We found that the replacement of the fourth point mutation in ho to the HO sequence restored HO activity to the chimeric endonuclease.
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PMID:Identification of the heterothallic mutation in HO-endonuclease of S. cerevisiae using HO/ho chimeric genes. 859 Apr 83

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.
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PMID:Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. 869 57

The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.
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PMID:Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease. 895 70

Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a site-specific double-stranded break at MAT, the yeast mating type locus. To identify other proteins involved in this process, we examined whether extracts prepared from ho- mutants contain additional factors that bind near the recognition sequence for Ho. Using an electrophoretic mobility shift assay, we isolated a chromatographic fraction that contains an activity, named YZbp, which binds to two sequences flanking the recognition sequence at MATalpha and to one sequence overlapping it at MATa. MAT plasmids carrying mutations in the YZbp recognition sequence are cleaved by purified Ho at wild-type efficiencies in an in vitro assay. These same plasmids, however, are not cleaved by Ho inside cells, demonstrating that YZbp acts as a positive activator of in vivo cleavage. YZbp is present in all cell types, even those not undergoing mating type switching, suggesting that it has additional cellular functions.
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PMID:Identification of a protein that binds to the Ho endonuclease recognition sequence at the yeast mating type locus. 900 Dec 31

Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a double-stranded DNA break at the yeast MAT locus. In this report, we characterize the fundamental biochemical properties of Ho. Using an assay that monitors cleavage of a MAT plasmid, we define an optimal in vitro reaction, showing in particular that the enzyme has a stringent requirement for zinc ions. This suggests that zinc finger motifs present in Ho are important for cleavage. The most unexpected feature of Ho, however, is its extreme inefficiency. Maximal cleavage occurs when Ho is present at a concentration of 1 molecule/3 base pairs of substrate DNA. Even under these conditions, complete digestion requires >2 h. This inefficiency results from two characteristics of Ho. First, Ho recycles slowly from cleaved product to new substrate, in part because the enzyme has an affinity for one end of its double strand break product. Second, high levels of cleavage in the in vitro reaction correlate with the appearance of large protein-DNA aggregates. At optimal Ho concentrations, these latter aggregates, referred to as "florettes," have an ordered structure consisting of a densely staining central region and loops of radiating DNA. These unusual properties may indicate that Ho plays a role in other aspects of mating type switching subsequent to double strand break formation.
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PMID:Ho endonuclease cleaves MAT DNA in vitro by an inefficient stoichiometric reaction mechanism. 905 34

Ho-endonuclease of the yeast, Saccharomyces cerevisiae, initiates a mating type switch by making a site-specific double strand break in the mating type gene, MAT. Ho is a dodecamer endonuclease and shares six of the seven intein motifs with PI- Sce I endonuclease, an intein encoded by the VMAI gene. We show that a 113 residue truncated Ho-endonuclease starting at intein motif C initiates a mating type switch in yeast. Ho is the only dodecamer endonuclease with zinc fingers. To see whether they have a role in determining site specificity we exchanged them for zinc fingers of the yeast transcription factor, Swi5. A chimeric endonuclease comprising the dodecamer motifs of Ho (C-E) and the zinc finger domain of Swi5 cleaves a Swi5 substrate plasmid in vivo. A similar chimera with the zinc fingers of SpI cleaves a GC box rich substrate plasmid. These experiments delineate a catalytic fragment of Ho-endonuclease that can be fused to various DNA binding moieties in the design of chimeric endonucleases with new site specificities.
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PMID:Targeting a truncated Ho-endonuclease of yeast to novel DNA sites with foreign zinc fingers. 946 31

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway.
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PMID:Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks. 987 Nov 17


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