Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

Biochemistry ◽  
1990 ◽  
Vol 29 (48) ◽  
pp. 10727-10733 ◽  
Author(s):  
John D. Taylor ◽  
Annette J. Goodall ◽  
Christian L. Vermote ◽  
Stephen E. Halford
Keyword(s):  
Dna Recognition ◽  
Modification System ◽  
Restriction Modification System ◽  
Restriction Modification

scholarly journals In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants

2008 ◽  
Vol 190 (24) ◽  
pp. 8003-8008 ◽  
Author(s):  
Edit Tímár ◽  
Pál Venetianer ◽  
Antal Kiss
Keyword(s):  
Restriction Endonuclease ◽  
Dna Methyltransferase ◽  
Term Survival ◽  
Modification System ◽  
Restriction Modification System ◽  
Low Concentrations ◽  
Compatible Plasmid ◽  
Restriction Modification

ABSTRACT The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.

scholarly journals Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 703
Author(s):  
Aistė Bulavaitė ◽  
Indre Dalgediene ◽  
Vilma Michailoviene ◽  
Milda Pleckaityte
Keyword(s):  
Polyclonal Antibodies ◽  
Gardnerella Vaginalis ◽  
Wild Type ◽  
Modification System ◽  
Restriction Modification System ◽  
Digestion Pattern ◽  
Foreign Dna ◽  
Restriction Modification ◽  
Rease Activity

Intensive horizontal gene transfer may generate diversity and heterogeneity within the genus Gardnerella. Restriction-modification (R-M) systems and CRISPR-Cas are the principal defense tools against foreign DNA in bacteria. Nearly half of the tested Gardnerella spp. isolates harbored the CRISPR-Cas system. Several putative R-M systems of Gardnerella spp. strains were identified in the REBASE database. However, there was no experimental evidence for restriction endonuclease (REase) activity in the isolates. We showed that G. vaginalis strain ATCC 14018 contains the REase R.Gva14018I, which recognizes GGCC and most probably generates blunt ends on cleavage. Bioinformatics evidence and the activity of recombinant methyltransferase M.Gva14018I in vivo indicate that ATCC 14018 possesses a HaeIII-like R-M system. The truncated R.Gva14018I-4 lacking the C-terminal region was expressed in Escherichia coli and displayed wild-type REase specificity. Polyclonal antibodies against R.Gva14018I-4 detected the wild-type REase in the cell lysate of ATCC 14018. The cofactor requirements for activity and bioinformatics analysis indicated that R.Gva14018I belongs to the PD-(D/E)XK family of REases. The REase-like activity was observed in 5 of 31 tested Gardnerella spp. strains, although none of these matched the DNA digestion pattern of R.Gva14018I.

scholarly journals The Methyltransferase from the LlaDII Restriction-Modification System Influences the Level of Expression of Its Own Gene

2004 ◽  
Vol 186 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Lisa Lystbæk Christensen ◽  
Jytte Josephsen
Keyword(s):  
Transcriptional Activity ◽  
Methylation Status ◽  
Transcriptional Level ◽  
Modification System ◽  
Restriction Modification System ◽  
Recognition Sites ◽  
Promoter Probe ◽  
Promoter Probe Vector ◽  
Restriction Modification

ABSTRACT The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P llaDIIM ) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P llaDIIR ) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P llaDIIM are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR, which appears to be constitutively expressed.

scholarly journals Class I DISARM provides anti-phage and anti-conjugation activity by unmethylated DNA recognition

2021 ◽  
Author(s):  
Cristian Aparicio-Maldonado ◽  
Gal Ofir ◽  
Andrea Salini ◽  
Rotem Sorek ◽  
Franklin L. Nobrega ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Dna Recognition ◽  
Modification System ◽  
Innate Defense ◽  
Restriction Modification System ◽  
Double Stranded Dna ◽  
Class 1 ◽  
Island System ◽  
Restriction Modification ◽  
Antiphage Activity

Bacteriophages impose a strong evolutionary pressure on microbes for the development of mechanisms of survival. Multiple new mechanisms of innate defense have been described recently, with the molecular mechanism of most of them remaining uncharacterized. Here, we show that a Class 1 DISARM (defense island system associated with restriction-modification) system from Serratia sp. provides broad protection from double-stranded DNA phages, and drives a population of single-stranded phages to extinction. We identify that protection is not abolished by deletion of individual DISARM genes and that the absence of methylase genes drmMI and drmMII does not result in autoimmunity. In addition to antiphage activity we also observe that DISARM limits conjugation, and this activity is linked to the number of methylase cognate sites in the plasmid. Overall, we show that Class 1 DISARM provides robust anti-phage and anti-plasmid protection mediated primarily by drmA and drmB, which provide resistance to invading nucleic acids using a mechanism enhanced by the recognition of unmethylated cognate sites of the two methylases drmMI and drmMII.

Cloning the KpnI restriction-modification system in Escherichia coli

Gene ◽  
1991 ◽  
Vol 97 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Alan W. Hammond ◽  
Gary F. Gerard ◽  
Deb K. Chatterjee
Keyword(s):  
Escherichia Coli ◽  
Modification System ◽  
Restriction Modification System ◽  
Restriction Modification

scholarly journals A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI)

2010 ◽  
Vol 38 (9) ◽  
pp. 3019-3030 ◽  
Author(s):  
Feroz Khan ◽  
Yoshikazu Furuta ◽  
Mikihiko Kawai ◽  
Katarzyna H. Kaminska ◽  
Ken Ishikawa ◽  
...  
Keyword(s):  
Mobile Genetic Element ◽  
Genetic Element ◽  
Modification System ◽  
Restriction Modification System ◽  
Restriction Modification

scholarly journals Complete Genome Sequence of Brevibacterium linens SMQ-1335

2016 ◽  
Vol 4 (6) ◽  
Author(s):  
Alessandra G. de Melo ◽  
Simon J. Labrie ◽  
Jeannot Dumaresq ◽  
Richard J. Roberts ◽  
Denise M. Tremblay ◽  
...  
Keyword(s):  
Complete Genome Sequence ◽  
Open Reading Frames ◽  
Type I ◽  
Industrial Strain ◽  
Modification System ◽  
Brevibacterium Linens ◽  
Restriction Modification System ◽  
New Type ◽  
Restriction Modification ◽  
Reading Frames

Brevibacterium linens is one of the main bacteria found in the smear of surface-ripened cheeses. The genome of the industrial strain SMQ-1335 was sequenced using PacBio. It has 4,209,935 bp, a 62.6% G+C content, 3,848 open reading frames, and 61 structural RNAs. A new type I restriction-modification system was identified.

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