Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease

Colleaux, Laurence; d'Auriol, L; Bétermier, Mireille; Cottarel, Guillaume; Jacquier, Alain; Galibert, Francis; Dujon, Bernard

doi:10.1016/0092-8674(86)90262-x

Cited by 261 publications

(141 citation statements)

References 29 publications

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“…This strain encodes the I-SceI restriction site (39,40) and expresses I-SceI from a xylose-inducible promoter to catalyze the formation of DSBs in vivo. We induced DSB formation in cells during active replication for 1 h and observed RecA-GFP foci in Ϸ90% of cells (Fig.…”

Section: The Subcellular Position Of Reca-gfp Foci Is Consistent Withmentioning

confidence: 99%

Replication is required for the RecA localization response to DNA damage in Bacillus subtilis

Simmons

Grossman

Walker

2007

Proc. Natl. Acad. Sci. U.S.A.

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In both prokaryotes and eukaryotes, proteins involved in DNA repair often organize into multicomponent complexes that can be visualized as foci in living cells. We used a RecA-GFP fusion to examine the subcellular cues that direct RecA-GFP to assemble as foci in response to DNA damage. We used two different methods to inhibit initiation of DNA replication and determined that DNA replication is required for the cell to establish RecA-GFP foci after exposure to DNA-damaging agents. Furthermore, use of endonuclease cleavage to generate a site-specific double-strand break demonstrated that the replication machinery (replisome) and DNA synthesis are required for assembly of RecA-GFP foci during repair of a double-strand break. We monitored the cellular levels of RecA and found that focus formation does not require further induction of protein levels, suggesting that foci result from a redistribution of existing protein to sites of damage encountered by the replisome. Taken together, our results support the model that existing RecA protein is recruited to ssDNA generated by the replisome at sites of DNA damage. These results provide insight into the mechanisms that the cell uses to recruit repair proteins to damaged DNA in living cells.replisome ͉ focus ͉ double-strand break

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Section: The Subcellular Position Of Reca-gfp Foci Is Consistent Withmentioning

confidence: 99%

Replication is required for the RecA localization response to DNA damage in Bacillus subtilis

Simmons

Grossman

Walker

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…One l of a solution freshly prepared by diluting an ethanolic solution of 1,10-phenanthroline (1 mM) and an aqueous cupric sulfate solution (0.23 mM) was added to 10 l of the appropriate free or I-SceI-bound DNA sample. Cleavage was initiated by the addition of 1 l of 58 mM MPA (final concentration, 5.8 mM), and the mixture was incubated at 23°C for either 30 s for OP 2 Cu ϩ or 2 min for 5 Phe OP 2 Cu ϩ . Cleavage was quenched by the addition of 1 l of 28 mM 2,9-dimethyl-orthophenanthroline (final concentration, 2.8 mM).…”

Section: Complex Probing Using Dna Cleavage Reagentsmentioning

confidence: 99%

“…We then used chemical probing agents to characterize the conformation of DNA in the complex. I-SceI protein-DNA complex was thus submitted to the nucleolytic attack of the cuprous complexes of 1,10-phenanthroline (OP 2 Cu ϩ and Phe-OP 2 Cu ϩ ), which results from the abstraction of C-1Ј hydrogen atom by a tetrahedral copper-oxo species bound within the minor groove (13,14,15,9) and to UVA/4Ј,5Ј-dihydro-7-methylpyrido [3,4-c]psoralen (DHMePyPs) photosensitization, which requires prior intercalation of the pyridopsoralen at selective 5Ј-TTA-3Ј sites (10). We also employed chemical modification agents of base and sugar residues (for a review, see Ref.…”

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confidence: 99%

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Chemical Probing Shows That the Intron-encoded Endonuclease I-SceI Distorts DNA through Binding in Monomeric Form to Its Homing Site

Beylot¹,

Spassky

2001

Journal of Biological Chemistry

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Despite its small size (27.6 kDa), the group I intronencoded I-SceI endonuclease initiates intron homing by recognizing and specifically cleaving a large intronless DNA sequence. Here, we used gel shift assays and footprinting experiments to analyze the interaction between I-SceI and its target. I-SceI was found to bind to its substrate in monomeric form. Footprinting using DNase I, hydroxyl radical, phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and base-modifying reagents revealed the asymmetric nature of the interaction and provided a first glimpse into the architecture of the complex. The protein interacts in the minor and major grooves and distorts DNA at three distinct sites: one at the intron insertion site and the other two, respectively, downstream (؊8, ؊9) and upstream (؉9, ؉10) from this site. The protein appears to stabilize the DNA curved around it by bridging the minor groove on one face of the helix. The scissile phosphates would lie on the outside of the bend, facing in the same direction relative to the DNA helical axis, as expected for an endonuclease that generates 3 overhangs. An internally consistent model is proposed in which the protein would take advantage of the concerted flexibility of the DNA sequence to induce a synergistic binding/kinking process, resulting in the correct positioning of the enzyme active site.I-SceI is a homing endonuclease encoded by the mobile group I intron of the large rRNA gene of Saccharomyces cerevisiae (1, 2). This family of enzymes mediates the propagation of the intron by cutting intronless genes at the site of intron insertion (reviewed in Ref.3). Like restriction enzymes, homing endonucleases cleave double-stranded DNA with high specificity in the presence of divalent metal ions. However, they differ from restriction endonucleases in their recognition properties and structures, as well as in their genomic location (4). In particular, whereas restriction enzymes have short recognition sequences (3-8 bp), 1 homing endonucleases, despite their small size, recognize long DNA sequences (12-40 bp). They have been classified into four families on the basis of both their sequence motifs and DNA cleavage mechanism (3). to cleave DNA within its recognition sequence and leaves a 4-bp overhang presenting a 3Ј-hydroxyl terminus (5, 6). The enzyme displays a low turnover, probably because of its strong affinity for one of the products of the cleavage reaction (7).The interaction of homing endonucleases with their substrates raises an interesting question common to all the gene-regulatory proteins, namely: how can a small protein specifically recognize and modify a long DNA sequence? Understanding the molecular basis of such a mechanism is essential for elucidating many aspects of cellular control and is a prerequisite in any rational drug design program. It is clearly established that local and global DNA structural features that are highly sequence-dependent, play a primary role in the dynamics of protein-DNA recognition (8). Sequence recognition would...

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“…This family of enzymes includes intron-homing endonucleases, such as I-SceI, I-SceII, I-SceIII, I-SceIV, and Endo.SceI from yeast (29,30,(33)(34)(35)(36)(37)(38). Also included in this group are endonucleases encoded by inteins (internal protein sequences that are cleaved from larger primary translation products), such as PI-SceI (31, 32).…”

Section: Discussionmentioning

confidence: 99%

Ho Endonuclease Cleaves MAT DNA in Vitro by an Inefficient Stoichiometric Reaction Mechanism

Jin

Binkowski

Simon

et al. 1997

Journal of Biological Chemistry

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Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a doublestranded 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.Genetic recombination is the intracellular process that moves DNA sequence information from one genomic location to another. A plethora of genetic and biochemical experiments, conducted primarily in prokaryotic and fungal systems, has led to a detailed understanding of the DNA intermediates that arise during this process. Examples of such intermediates include broken duplexes, heteroduplex DNA joints, and Holliday junctions. The first of these intermediates (broken duplexes or double-stranded breaks (DSBs) 1 ) are thought to initiate recombination. Thus, in the yeast Saccharomyces cerevisiae, an organism in which DSB formation has been intensively analyzed, the following lines of evidence indicate that DSBs initiate meiotic recombination. First, DSBs are repaired using gene products required for meiotic recombination (reviewed in Ref. 1). Second, certain meiotic segregation patterns of yeast genes are consistent with molecular models of recombination that posit a DSB initiation event (2). Third, DSBs occur at regions of the genome defined genetically as initiators of recombination (3-11). Fourth, DSBs introduced into DNA during meiosis stimulate nearby meiotic recombination (12). Finally, meiotic DSBs precede and are independent of pairing between homologs, implying that DSBs initiate chromosomal pairing (13,14 (Fig. 1A). The W, X, Z1, and Z2 blocks are identical in a and ␣ cells, but each mating type contains a unique MAT Y sequence: Ya in a cells and Y␣ in ␣ cells. Roughly once per cell generation, new Ya or Y␣ information transposes into MAT from two unlinked and transcriptionally inactive loci known as HML␣ and HMRa, thereby switching the cell's mating ty...

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Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease

Cited by 261 publications

References 29 publications

Replication is required for the RecA localization response to DNA damage in Bacillus subtilis

Replication is required for the RecA localization response to DNA damage in Bacillus subtilis

Chemical Probing Shows That the Intron-encoded Endonuclease I-SceI Distorts DNA through Binding in Monomeric Form to Its Homing Site

Ho Endonuclease Cleaves MAT DNA in Vitro by an Inefficient Stoichiometric Reaction Mechanism

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