scholarly journals The Influence of 5′R and 5′S cdA and cdG on the Activity of BsmAI and SspI Restriction Enzymes

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3750
Author(s):  
Michał Szewczuk ◽  
Karolina Boguszewska ◽  
Julia Kaźmierczak-Barańska ◽  
Bolesław T. Karwowski
Keyword(s):  
Genetic Material ◽  
Restriction Enzymes ◽  
Dna Lesions ◽  
Recognition Site ◽  
Recognition Sequence ◽  
Cellular Mechanisms ◽  
Modification System ◽  
Restriction Modification System ◽  
Restriction Modification ◽  
Oligonucleotide Sequence

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5′,8-cyclo-2′-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction–modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme’s recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5′S)-5′,8-cyclo-2′-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5′R)-5′,8-cyclo-2′-deoxyadenosine (RcdA). Interestingly, in the case of 5′,8-cyclo-2′-deoxyguanosine (cdG), both 5′S and 5′R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.

scholarly journals Lactococcal Plasmid pNP40 Encodes a Novel, Temperature-Sensitive Restriction-Modification System

2004 ◽  
Vol 70 (9) ◽  
pp. 5546-5556 ◽  
Author(s):  
Jonathan O'Driscoll ◽  
Frances Glynn ◽  
Oonagh Cahalane ◽  
Mary O'Connell-Motherway ◽  
Gerald F. Fitzgerald ◽  
...  
Keyword(s):  
Restriction Enzymes ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Modification System ◽  
Restriction Modification System ◽  
Naturally Occurring ◽  
Low Copy Number ◽  
Encoding Genes ◽  
Restriction Modification ◽  
Specific Mrna

ABSTRACT A novel restriction-modification system, designated LlaJI, was identified on pNP40, a naturally occurring 65-kb plasmid from Lactococcus lactis. The system comprises four adjacent similarly oriented genes that are predicted to encode two m5C methylases and two restriction endonucleases. The LlaJI system, when cloned into a low-copy-number vector, was shown to confer resistance against representatives of the three most common lactococcal phage species. This phage resistance phenotype was found to be strongly temperature dependent, being most effective at 19°C. A functional analysis confirmed that the predicted methylase-encoding genes, llaJIM1 and llaJIM2, were both required to mediate complete methylation, while the assumed restriction enzymes, specified by llaJIR1 and llaJIR2, were both necessary for the complete restriction phenotype. A Northern blot analysis revealed that the four LlaJI genes are part of a 6-kb operon and that the relative abundance of the LlaJI-specific mRNA in the cells does not appear to contribute to the observed temperature-sensitive profile. This was substantiated by use of a LlaJI promoter-lacZ fusion, which further revealed that the LlaJI operon appears to be subject to transcriptional regulation by an as yet unidentified element(s) encoded by pNP40.

scholarly journals LraI fromLactococcus raffinolactisBGTRK10-1, an Isoschizomer ofEcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Marija Miljkovic ◽  
Milka Malesevic ◽  
Brankica Filipic ◽  
Goran Vukotic ◽  
Milan Kojic
Keyword(s):  
Restriction Enzyme ◽  
Specific Activity ◽  
Restriction Enzymes ◽  
Ion Concentration ◽  
Gene Encoding ◽  
Modification System ◽  
Star Activity ◽  
Restriction Modification System ◽  
Monovalent Ions ◽  
Restriction Modification

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity.Lactococcus raffinolactisBGTRK10-1 expressesLraI Type II restriction-modification enzyme, whose activity is similar to that shown forEcoRI;LraI methyltransferase protects DNA fromEcoRI cleavage. The gene encodingLraI endonuclease was cloned and overexpressed inE. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at −20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity ofLraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence forLraI restriction enzyme was determined as 5′-G/AATTC-3′, indicating thatLraI restriction enzyme is an isoschizomer ofEcoRI. In the reaction buffer with a lower salt concentration,LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5′-A/AATTC-3′, leaving the same sticky ends on fragments asEcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin ofLraI restriction-modification system with previously describedEcoRI-like restriction-modification systems.

scholarly journals Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a PyrococcusStrain and Cloning of the PspGI Restriction-Modification System in Escherichia coli

1998 ◽  
Vol 64 (10) ◽  
pp. 3669-3673 ◽  
Author(s):  
Richard Morgan ◽  
Jian-ping Xiao ◽  
Shuang-yong Xu
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Expression System ◽  
Sequence Motifs ◽  
Recognition Sequence ◽  
Modification System ◽  
Conserved Sequence ◽  
Restriction Modification System ◽  
Diagnostic Applications ◽  
Restriction Modification

ABSTRACT An extremely thermostable restriction endonuclease,PspGI, was purified from Pyrococcus sp. strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5′ ^CCWGG 3′ (W is A or T). PspGI digestion can be carried out at 65 to 85°C. To express PspGI at high levels, the PspGI restriction-modification genes (pspGIR andpspGIM) were cloned in Escherichia coli. M.PspGI contains the conserved sequence motifs of α-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI fromMicrococcus variabilis. In a segment of 87 amino acid residues, PspGI shows significant sequence similarity toEcoRII and to regions of SsoII andStyD4I which have a closely related recognition sequence (5′ ^CCNGG 3′). PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95°C. PspGI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.

scholarly journals Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Fuzhou Ye ◽  
Ioly Kotta-Loizou ◽  
Milija Jovanovic ◽  
Xiaojiao Liu ◽  
David TF Dryden ◽  
...  
Keyword(s):  
Restriction Enzymes ◽  
Structural Basis ◽  
Bacteriophage T7 ◽  
Transcription Inhibition ◽  
Modification System ◽  
Host Restriction ◽  
Restriction Modification System ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
Restriction Modification

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.

scholarly journals Distribution of the SsuDAT1I Restriction-Modification System among Different Serotypes ofStreptococcus suis

2001 ◽  
Vol 183 (18) ◽  
pp. 5436-5440 ◽  
Author(s):  
Tsutomu Sekizaki ◽  
Makoto Osaki ◽  
Daisuke Takamatsu ◽  
Yoshihiro Shimoji
Keyword(s):  
Genetic Divergence ◽  
Field Isolate ◽  
Gene Sequences ◽  
M Gene ◽  
Recognition Sequence ◽  
Modification System ◽  
Restriction Modification System ◽  
Serotype 1 ◽  
Reference Strains ◽  
Restriction Modification

ABSTRACT The SsuDAT1I restriction-modification (R-M) system, which contains two methyltransferases and two restriction endonucleases with recognition sequence 5′-GATC-3′, was first found in a field isolate of Streptococcus suis serotype 2. Isoschizomers of the R-M system were found in the same locus betweenpurH and purD in a field isolate of serotype 1/2 and the reference strains of serotypes 3, 7, 23, and 26 among 29 strains of different serotypes examined in this study. The R-M gene sequences in serotypes 1/2, 3, 7, and 23 were very similar to those of SsuDAT1I, whereas those in serotype 26 were less similar. These results indicate intraspecies recombination among them and genetic divergence through their evolution.

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.

Sign in / Sign up

Export Citation Format

Share Document