DNA cleavage by type III restriction-modification enzyme Eco P15I is independent of spacer distance between two head to head oriented recognition sites †,1 †In memory of Dieter Cech (1944–1996), Professor of bioorganic chemistry at the Humboldt University, Berlin. 1Edited by J. Karn

2001 ◽  
Vol 312 (4) ◽  
pp. 687-698 ◽  
Author(s):  
Merlind Mücke ◽  
Stefanie Reich ◽  
Elisabeth Möncke-Buchner ◽  
Monika Reuter ◽  
Detlev H Krüger
Keyword(s):  
Bioorganic Chemistry ◽  
Dna Cleavage ◽  
Type Iii ◽  
Recognition Sites ◽  
Humboldt University ◽  
Restriction Modification ◽  
Modification Enzyme

scholarly journals DNA-mediated coupling of ATPase, translocase and nuclease activities of a Type ISP restriction-modification enzyme

2020 ◽  
Vol 48 (5) ◽  
pp. 2594-2603
Author(s):  
Mahesh Kumar Chand ◽  
Vanessa Carle ◽  
K G Anuvind ◽  
Kayarat Saikrishnan
Keyword(s):  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Target Sequence ◽  
Dna Translocation ◽  
Wild Type Enzyme ◽  
Functional Activities ◽  
Dna Cleavage Activity ◽  
Stage Process ◽  
Restriction Modification ◽  
Modification Enzyme

Abstract Enzymes involved in nucleic acid transactions often have a helicase-like ATPase coordinating and driving their functional activities, but our understanding of the mechanistic details of their coordination is limited. For example, DNA cleavage by the antiphage defense system Type ISP restriction-modification enzyme requires convergence of two such enzymes that are actively translocating on DNA powered by Superfamily 2 ATPases. The ATPase is activated when the enzyme recognizes a DNA target sequence. Here, we show that the activation is a two-stage process of partial ATPase stimulation upon recognition of the target sequence by the methyltransferase and the target recognition domains, and complete stimulation that additionally requires the DNA to interact with the ATPase domain. Mutagenesis revealed that a β-hairpin loop and motif V of the ATPase couples DNA translocation to ATP hydrolysis. Deletion of the loop inhibited translocation, while mutation of motif V slowed the rate of translocation. Both the mutations inhibited the double-strand (ds) DNA cleavage activity of the enzyme. However, a translocating motif V mutant cleaved dsDNA on encountering a translocating wild-type enzyme. Based on these results, we conclude that the ATPase-driven translocation not only brings two nucleases spatially close to catalyze dsDNA break, but that the rate of translocation influences dsDNA cleavage.

Type III restriction enzymes need two inversely oriented recognition sites for DNA cleavage

Nature ◽  
1992 ◽  
Vol 355 (6359) ◽  
pp. 467-469 ◽  
Author(s):  
Andreas Meisel ◽  
Thomas A. Bickle ◽  
Detlev H. Kriiger ◽  
Cornelia Schroeder
Keyword(s):  
Dna Cleavage ◽  
Restriction Enzymes ◽  
Type Iii ◽  
Recognition Sites

scholarly journals DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI

2009 ◽  
Vol 37 (21) ◽  
pp. 7206-7218 ◽  
Author(s):  
Rachel M. Smith ◽  
Fiona M. Diffin ◽  
Nigel J. Savery ◽  
Jytte Josephsen ◽  
Mark D. Szczelkun
Keyword(s):  
Dna Cleavage ◽  
Single Polypeptide ◽  
Restriction Modification ◽  
Modification Enzyme

scholarly journals Type III restriction-modification enzymes: a historical perspective

2013 ◽  
Vol 42 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Desirazu N. Rao ◽  
David T. F. Dryden ◽  
Shivakumara Bheemanaik
Keyword(s):  
Historical Perspective ◽  
Type Iii ◽  
Restriction Modification

scholarly journals A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency inCampylobacter jejuni

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Ximin Zeng ◽  
Zuowei Wu ◽  
Qijing Zhang ◽  
Jun Lin
Keyword(s):  
Comparative Genomics ◽  
Heat Shock ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Campylobacter Jejuni ◽  
Selection Marker ◽  
Sequencing Analysis ◽  
Content Type ◽  
Restriction Modification ◽  
Modification Enzyme

ABSTRACTConjugation is an important mechanism for horizontal gene transfer inCampylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency inCampylobacterspp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC)C. jejunistrain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFCC. jejunistrain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFCC. jejunistrain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (akaCjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFCC. jejunistrains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest theEscherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency inC. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency inC. jejuni.IMPORTANCEConjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance.Campylobacter jejuni, the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency inC. jejuniare still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency inC. jejuni. The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.

scholarly journals Stability of EcoRI Restriction-Modification Enzymes In Vivo Differentiates the EcoRI Restriction-Modification System from Other Postsegregational Cell Killing Systems

2005 ◽  
Vol 187 (19) ◽  
pp. 6612-6621 ◽  
Author(s):  
Asao Ichige ◽  
Ichizo Kobayashi
Keyword(s):  
Restriction Enzyme ◽  
Cell Killing ◽  
Host Cells ◽  
Gene System ◽  
Ecori Restriction ◽  
Differential Stability ◽  
Postsegregational Killing ◽  
Restriction Modification ◽  
Modification Enzyme

ABSTRACT Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.

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