Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism

Salaj-Šmic, Erika; Maršić, Nataša; Trgovčević, Željko; Lloyd, Robert G.

doi:10.1128/jb.179.6.1852-1856.1997

Cited by 9 publications

(9 citation statements)

References 44 publications

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“…These in vitro results are consistent with previous in vivo data demonstrating a reduction in restriction by EcoKI in the absence of RecBCD ( 33 ). Although the reason for this was unclear at the time, our data suggest that the reduction may at least be due in part to the accumulation of competitor DNA resulting from DNA cleavage by EcoKI.…”

Section: Discussionsupporting

confidence: 93%

“…The observation of catalytic function for type I REs alters the in vivo mechanistic view of these previously enigmatic enzymes. The idea that these enzymes might remain bound to restricted phage DNA suggests a requirement for their rapid removal by either the action of exonucleases such as RecBCD or ExoIII ( 11 , 33 , 34 ) or by proteases ( 37–39 ). Failure to remove type I restriction enzymes from the DNA using the abovementioned, ATP-requiring processes either separately, or in combination, could prove costly to a cell.…”

Section: Discussionmentioning

confidence: 99%

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Type I restriction endonucleases are true catalytic enzymes

Bianco¹,

Xu²,

Chi³

2009

Nucleic Acids Research

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Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Type I restriction endonucleases are true catalytic enzymes

Bianco¹,

Xu²,

Chi³

2009

Nucleic Acids Research

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“…The outcome of this activity is that the point of DNA cleavage is random and the resultant fragments are of highly variable size (79). It is interesting that the presence of a Type I Restriction−Modification system increases the frequency of recombination in E. coli (84)(85), which may provide some insight into why random cleavage is a useful strategy and supports the observation described above that the DNA ends of these random sized fragments are accessible to recombination enzymes (86-87).…”

Section: Dna Translocation and Molecular Motor Activitysupporting

confidence: 53%

Mechanistic insight into Type I restriction endonucleases

Youell

Firman²

2012

Front Biosci

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Restriction and modification are two opposing activities that are used to protect bacteria from cellular invasion by DNA (e.g. bacteriophage infection). Restriction activity involves cleavage of the DNA; while modification activity is the mechanism used to "mark" host DNA and involves DNA methylation. The study of Type I restriction enzymes has often been seen as an esoteric exercise and this reflects some of their more unusual properties - non-stoichiometric (non-catalytic) cleavage of the DNA substrate, random cleavage of DNA, a massive ATPase activity, and the ability to both cleave DNA and methylate DNA. Yet these enzymes have been found in many bacteria and are very efficient as a means of protecting bacteria against bacteriophage infection, indicating they are successful enzymes. In this review, we summarise recent work on the mechanisms of action, describe switching of function and review their mechanism of action. We also discuss structural rearrangements and cellular localisation, which provide powerful mechanisms for controlling the enzyme activity. Finally, we speculate as to their involvement in recombination and discuss their relationship to helicase enzymes.

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“…The strain E. coli C600r k À m k À , which lacks the EcoKI restriction-modification system, was used for preparation of unmodified phage stock lvirC (designated as lvir.0) (Salaj-Šmic et al 1997). Phages lvirC, grown on the E. coli AB1157 (r k 1 m k 1 ) strain, are modified and designated lvir.K.…”

Section: Bacterial Strains and Bacteriophagesmentioning

confidence: 99%

Roles of PriA Protein and Double-Strand DNA Break Repair Functions in UV-Induced Restriction Alleviation inEscherichia coli

et al. 2006

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It has been widely considered that DNA modification protects the chromosome of bacteria E. coli K-12 against their own restriction-modification systems. Chromosomal DNA is protected from degradation by methylation of target sequences. However, when unmethylated target sequences are generated in the host chromosome, the endonuclease activity of the EcoKI restriction-modification enzyme is inactivated by the ClpXP protease and DNA is protected. This process is known as restriction alleviation (RA) and it can be induced by UV irradiation (UV-induced RA). It has been proposed that chromosomal unmethylated target sequences, a signal for the cell to protect its own DNA, can be generated by homologous recombination during the repair of damaged DNA. In this study, we wanted to further investigate the genetic requirements for recombination proteins involved in the generation of unmethylated target sequences. For this purpose, we monitored the alleviation of EcoKI restriction by measuring the survival of unmodified l in UV-irradiated cells. Our genetic analysis showed that UV-induced RA is dependent on the excision repair protein UvrA, the RecA-loading activity of the RecBCD enzyme, and the primosome assembly activity of the PriA helicase and is partially dependent on RecFOR proteins. On the basis of our results, we propose that unmethylated target sequences are generated at the D-loop by the strand exchange of two hemi-methylated duplex DNAs and subsequent initiation of DNA replication.

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Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism

Cited by 9 publications

References 44 publications

Type I restriction endonucleases are true catalytic enzymes

Type I restriction endonucleases are true catalytic enzymes

Mechanistic insight into Type I restriction endonucleases

Roles of PriA Protein and Double-Strand DNA Break Repair Functions in UV-Induced Restriction Alleviation inEscherichia coli

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