Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease.

Heitman, Joseph; Zinder, Norton D.; Model, Peter

doi:10.1073/pnas.86.7.2281

Cited by 89 publications

(58 citation statements)

References 33 publications

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“…However, when a restriction endonuclease exhibits star activity in vitro, a question that always arises is how the bacterial cell protects its DNA from the cleavage at star sites. It has been proposed that either the cognate methyltransferase can methylate star sites in vivo (24), or the cell has an efficient repair system that can seal the strand breaks produced at star sites (25,26). For SgrAI, canonical site methylation should abolish the production of the SgrAI-termini required for secondary-site cleavage, so that secondary sites, even when unmethylated, would be much more resistant to SgrAI.…”

Section: Discussionmentioning

confidence: 99%

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Bitinaite

Schildkraut²

2002

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

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The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5 -CPu2CCGGPyG. Previous work had indicated that SgrAI must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999) J. Biol. Chem. 274, 36379 -36386]. In the present study, SgrAI is shown to cleave not only its canonical sequences, but also the sequences 5 -CPuCCGGPy(A,T,C) and 5 -CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7, SgrAI cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for SgrAI assist in the cleavage of secondary sites. The SgrAI-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAI-termini over substrate, the SgrAI specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which SgrAI utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the SgrAI restriction endonuclease is proposed.

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Section: Discussionmentioning

confidence: 99%

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Bitinaite

Schildkraut²

2002

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

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“…Our selfish gene hypothesis suggests that bacteria may have evolved anti-RM systems as their viruses have done. We can think of four possible anti-RM mechanisms: (i) methyl-specific restriction systems (37,38); (ii) methylation by bacterial solo methylases (39), which may protect bacterial chromosomes from postsegregational cleavage by certain RM systems; (iii) homologous recombination (40); and (iv) ligation with DNA ligase (41).…”

Section: Discussionmentioning

confidence: 99%

Restriction-modification systems as genomic parasites in competition for specific sequences.

Kusano

Naito

Handa

et al. 1995

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

102

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Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virusinfected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.A type II restriction endonuclease makes a double-strand break within or near a specific recognition sequence in duplex DNA. A cognate modification enzyme methylates the recognition sequence to protect it from the cleavage (1, 2). It is widely accepted that the evolution and maintenance of restriction-modification (RM) systems have been driven by the protection from foreign DNA that they afford to cells. The RM systems do protect cells from infection with some viruses by cleaving their DNA (for example, see ref.3) and are likely to be responsible both for the evolution of antirestriction mechanisms and for the paucity of some restriction sites in certain viruses and plasmids (4).Recent experimental and theoretical analyses (5, 6), however, seem to us to bring into question the efficacy of virusmediated selection for RM systems. Defense by RM systems is short-lived because invading viral DNA will occasionally escape restriction and will become modified, thus affording protection from restriction to itself and its descendants (5, 6). Bacteria will more likely develop other, longer-lasting means of resistance to viruses, such as alterations in the receptor required for infection (5, 6). Although RM systems can provide bacteria with advantage when they are invading new habitats full of phages, it is not clear whether such colonization selection is realistic under natural conditions (5, 6).It is also unclear whether the above "cellular defense" hypothesis can account for the following properties of type II RM systems (1, 2). (i) Their individual high specificity and collective wide diversity in the sequence recognition. (ii) The tight linkage of cognate restriction and modification ...

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“…--Galactosidase expression is increased upon treatment of the cells with UV, mitomycin C, or other DNA-damaging agents (5-8, 12, 21). It was shown that EcoRI temperature-sensitive mutants or EcoRI mutants with partial cleavage activity induce the SOS response by causing damage to the E. coli chromosome (5,7,8). Furthermore, EcoRI mutants with relaxed specificity have been isolated by screening for SOS-inducible blue phenotype in the presence of EcoRI methylase (6).…”

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confidence: 99%

Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

Schildkraut

1991

J Bacteriol

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A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 103-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2, in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherirhia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were 'located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2Y as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metai preference a$ well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.The BamHI restriction endonuclease purified from Bacillus amyloliquefaciens H cleaves the symmetric DNA sequence 5'-GGATCC-3' between the two guanines on both strands (reviewed in reference 17). The coding sequence for BamHI has been cloned and sequenced, and the enzyme has been overexpressed in Escherichia coli (2,3,10). Like all type II restriction endonucleases, BamHI requires the divalent cation Mg2' as a cofactor for cleavage activity. Mn2+ can be substituted for Mg2+ in the reaction, but the enzyme activity is reduced (24). Mn2+ also decreases the enzyme specificity, allowing BamHI to cleave noncanonical sequences (BamHI star sites; 10, 14). The DNA-binding activity, however, is independent of metal cofactor (17, 28).The SOS regulon consisting of about 20 genes in E. coli is negatively regulated by the LexA repressor (reviewed in references 19, 22, and 25). The repressor can be cleaved and inactivated by the RecA protein to derepress the genes in the regulon. The proteolytic activity of the RecA protein is activated by a signal (e.g., single-stranded DNA or oligonucleotides) upon DNA damage or inhibition of DNA replication. As the result of DNA damage or DNA replication interference, the genes in the SOS regulon are expressed at an increased level. E. coli indicator strain AP1-200 contains a dinD: :lacZ fusion in which the lactose operon is fused to a DNA damage-inducible promoter (12). --Galactosidase expression is increased upon treatment of the cells with UV, mitomycin...

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Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease.

Cited by 89 publications

References 33 publications

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Restriction-modification systems as genomic parasites in competition for specific sequences.

Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

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