Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs.

Webb, Julie L.; King, Gareth; Ternent, Diane; Titheradge, Annette J. B.; Murray, Noreen E.

doi:10.1002/j.1460-2075.1996.tb00551.x

Cited by 58 publications

(52 citation statements)

References 38 publications

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“…Bacterial strains are listed in Table 1. Integration-deficient, hsdcI857 phages were used to transfer hsd alleles to bacterial chromosomes: NM1367 includes hsd⌬RM(F269G)S ϩ ; NM1376, hsdM ϩ S ϩ ; NM1394, hsdM(F269G)S ϩ ; and NM1384, hsdR(A619V) (17). JC9935 was used as the donor of the following derivatives of FЈ101:…”

Section: Methodsmentioning

confidence: 99%

Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

Makovets

Doronina

Murray

1999

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

152

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ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd ؉ bacterium as the result of replication errors or recombinationdependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with Ϸ600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.Within a bacterium that has a classical restriction and modification (R-M) system, the nucleotide sequences that define the targets for attack by the resident restriction endonuclease are concealed by the modification of appropriate bases within them. For some systems this modification is achieved by the methylation of specific adenine residues, and for others it is achieved by methylation of cytosine residues. The restriction endonuclease has the potential to attack DNA from different strains of the same species because foreign DNA generally lacks the protective imprint of the relevant methyltransferase (for reviews see refs. 1 and 2). Restriction of the host cell's newly synthesized DNA normally is avoided, because the unmethylated strand of each target sequence produced by DNA replication is methylated before the next round of replication. If, however, resident DNA were to acquire unmodified target sequences, would it, like foreign DNA, become a substrate for restriction? In this paper we show that in situations where the modification of the host DNA by a type I R-M system fails, an alternative level of protection impairs the endonuclease activity of the restriction system and the bacteria survive.A type I R-M system is encoded by three genes: hsdR, hsdM, and hsdS. The three polypeptides, HsdR, HsdM, and HsdS, often designated R, M, and S, assemble to give an enzyme (R 2 M 2 S 1 ) that modifies hemimethylated DNA and restricts unmethylated DNA. A smaller complex (M 2 S 1 ) has only the methyltransferase activity. The S subunit confers target specificity; hence, both complexes and both activities respond to the same nucleotide sequence.Type

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Section: Methodsmentioning

confidence: 99%

Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

Makovets

Doronina

Murray

1999

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

152

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“…D364 was alternately grown on the EcoKI-containing strain IJ891 (E. coli K-12 ⌬lacX74 thi) and the isogenic nonrestricting ⌬hsd strain IJ1133 [IJ891 ⌬(mcrC-mrr)::Tn10] to enrich for mutants that have lost EcoKI recognition sites. After several passages, phages then were propagated alternately on IJ1133 and on IJ891 containing pSB2, a plasmid that overexpresses hsdR (8), to increase the selection pressure for loss of EcoKI sites. A two-step procedure was necessary, because D364 does not plate at detectable frequencies on IJ891(pSB2).…”

Section: Methodsmentioning

confidence: 99%

“…A current model for cleavage site selection, determined from in vitro experiments, posits that cutting occurs when two translocating EcoKI molecules collide (7). The observation that mutants containing two recognition sites for EcoKI are restricted 15-fold more efficiently than expected if two enzyme molecules cut independently provides in vivo support for the model (8).…”

mentioning

confidence: 99%

Translocation and specific cleavage of bacteriophage T7 DNA in vivo by Eco KI

García

Molineux

1999

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

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Infection of Escherichia coli containing the type I restriction enzymeEcoKI by bacteriophage T7 0.3 mutants leads to restriction during the late stages of genome entry and during DNA replication. Patterns of cleavage in vivo suggest that some cutting occurs near the midpoint of two recognition sites, consistent with the idea that EcoKI translocates DNA bidirectionally through itself and cuts when two enzyme molecules collide. Rapid ejection of a 0.3 ؉ T7 genome from a bacteriophage particle results in degradation of the infecting DNA by EcoKI, showing that the normal T7 DNA translocation process delays restriction. A unique recognition site inserted at the genomic left end allows EcoKI to function as a molecular motor and to translocate the remaining 39 kilobases of T7 DNA into the cell.T ype I restriction-modification systems of Escherichia coli serve to protect the bacterium from many phages (1). E. coli K-12 strains harboring the hsd locus contain the enzyme EcoKI, a multisubunit enzyme consisting of a specificity subunit and two each of restriction and modification subunits (2). EcoKI binds S-adenosylmethionine forming an activated enzyme (3), which binds the DNA sequence 5Ј-AACN 6 GTGC (4) and determines its methylation state. If the recognition sequence is nonmethylated, EcoKI hydrolyzes ATP and spools flanking DNA through itself (5); because it remains bound to its recognition sequence, highly twisted DNA loops are formed (6). A current model for cleavage site selection, determined from in vitro experiments, posits that cutting occurs when two translocating EcoKI molecules collide (7). The observation that mutants containing two recognition sites for EcoKI are restricted 15-fold more efficiently than expected if two enzyme molecules cut independently provides in vivo support for the model (8).Many phages and conjugal plasmids escape restriction by type I restriction-modification systems (1, 9). Some phage genomes contain exotically modified bases (10); some plasmids and the phages T7 and T3 synthesize a direct inhibitor of type I restriction-modification systems (11-13). Inhibition of type I restriction by T7 requires stoichiometric amounts of the early protein gp0.3, which binds to EcoKI and prevents it from binding target DNA (14); gp0.3 also obstructs DNA cleavage and methylation after EcoKI has interacted with its recognition sequence (15). Escape of T7 DNA from EcoKI restriction in vivo depends on the synthesis of sufficient gp0.3 before the viral genome becomes cleaved. T7 0.3 mutants containing unmodified DNA plate on hosts harboring EcoKI at an efficiency of 10 Ϫ2 to 10 Ϫ4 (12), somewhat higher but similar to the efficiency of unmodified . Nevertheless, stocks of T7 0.3 mutants can be made on restricting cells, although lysates have low titers and progeny genomes are modified only partially. It was suggested that the incomplete restriction and modification of T7 0.3 DNA was caused by the rapid replication phase and short latent period of the phage.Cellular internalization of T7 DNA is coupled to t...

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“…Nearly all reported mutations affecting DEAD-box motifs impair the hydrolysis of NTP or the coupling of NTP hydrolysis to nucleotide unwinding (64a). Mutagenesis of the hsdR gene of EcoKI showed that each of the seven DEAD-box motifs is essential for restriction in vivo (35,188).…”

Section: Restriction Subunit-hsdrmentioning

confidence: 99%

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

Murray

2000

Microbiol Mol Biol Rev

406

481

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SUMMARY Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.

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Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs.

Cited by 58 publications

References 38 publications

Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

Translocation and specific cleavage of bacteriophage T7 DNA in vivo by Eco KI

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

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