scholarly journals The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

2017 ◽  
Author(s):  
Vitali Bialevich ◽  
Dhiraj Sinha ◽  
Katsiaryna Shamayeva ◽  
Alena Guzanova ◽  
David Řeha ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Cleavage ◽  
Contact Region ◽  
Large Subunit ◽  
Helicase Domain ◽  
Type I ◽  
Dna Translocation ◽  
Helical Domain ◽  
Restriction Modification

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types, HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

scholarly journals The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2887 ◽  
Author(s):  
Vitali Bialevich ◽  
Dhiraj Sinha ◽  
Katsiaryna Shamayeva ◽  
Alena Guzanova ◽  
David Řeha ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Cleavage ◽  
Contact Region ◽  
Large Subunit ◽  
Helicase Domain ◽  
Type I ◽  
Dna Translocation ◽  
Helical Domain ◽  
Restriction Modification

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes usingin vivoandin vitroexperiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

scholarly journals The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

2017 ◽  
Author(s):  
Vitali Bialevich ◽  
Dhiraj Sinha ◽  
Katsiaryna Shamayeva ◽  
Alena Guzanova ◽  
David Řeha ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Cleavage ◽  
Contact Region ◽  
Large Subunit ◽  
Helicase Domain ◽  
Type I ◽  
Dna Translocation ◽  
Helical Domain ◽  
Restriction Modification

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types, HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

2004 ◽  
Vol 11 (9) ◽  
pp. 838-843 ◽  
Author(s):  
Ralf Seidel ◽  
John van Noort ◽  
Carsten van der Scheer ◽  
Joost G P Bloom ◽  
Nynke H Dekker ◽  
...  
Keyword(s):  
Real Time ◽  
Type I ◽  
Dna Translocation ◽  
Time Observation ◽  
Restriction Modification ◽  
Real Time Observation ◽  
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.

scholarly journals Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis

2020 ◽  
Vol 48 (19) ◽  
pp. 11040-11053
Author(s):  
Nadav Ben-Assa ◽  
Michael J Coyne ◽  
Alexey Fomenkov ◽  
Jonathan Livny ◽  
William P Robins ◽  
...  
Keyword(s):  
Bacteroides Fragilis ◽  
Surface Proteins ◽  
Type I ◽  
Phase Variable ◽  
Modification System ◽  
Restriction Modification System ◽  
Recognition Sites ◽  
Specificity Protein ◽  
Restriction Modification

Abstract The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

scholarly journals Structural basis for inhibition of an archaeal CRISPR–Cas type I-D large subunit by an anti-CRISPR protein

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Cemre Manav ◽  
Lan B. Van ◽  
Jinzhong Lin ◽  
Anders Fuglsang ◽  
Xu Peng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Functional State ◽  
Dna Cleavage ◽  
Large Subunit ◽  
Domain Architecture ◽  
Structural Basis ◽  
Type I ◽  
Helicase Activity ◽  
Nuclease Domain ◽  
Sulfolobus Islandicus

AbstractA hallmark of type I CRISPR–Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3’ subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR–Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

scholarly journals Translocation, switching and gating: potential roles for ATP in long-range communication on DNA by Type III restriction endonucleases

2011 ◽  
Vol 39 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Mark D. Szczelkun
Keyword(s):  
Long Range ◽  
Atp Hydrolysis ◽  
Helicase Domain ◽  
Dna Translocation ◽  
Base Pairs ◽  
Range Interaction ◽  
Type Iii ◽  
Long Range Interaction ◽  
Atp Binding And Hydrolysis ◽  
Restriction Modification

To cleave DNA, the Type III RM (restriction–modification) enzymes must communicate the relative orientation of two recognition sequences, which may be separated by many thousands of base pairs. This long-range interaction requires ATP hydrolysis by a helicase domain, and both active (DNA translocation) and passive (DNA sliding) modes of motion along DNA have been proposed. Potential roles for ATP binding and hydrolysis by the helicase domains are discussed, with a focus on bipartite ATPases that act as molecular switches.

scholarly journals A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

2018 ◽  
Author(s):  
Senén D. Mendoza ◽  
Joel D. Berry ◽  
Eliza S. Nieweglowska ◽  
Lina M. Leon ◽  
David A. Agard ◽  
...  
Keyword(s):  
Type I ◽  
Type Ii ◽  
Immune Systems ◽  
Dna Targeting ◽  
Rna Targeting ◽  
Immune Pathways ◽  
Type V ◽  
Restriction Modification

All viruses require strategies to inhibit or evade the immunity pathways of cells they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid nucleic-acid targeting immune pathways such as CRISPR-Cas and restriction endonucleases to replicate efficiently1. Here, we show that a jumbo phage infecting Pseudomonas aeruginosa, phage ΦKZ, is resistant to many immune systems in vivo, including CRISPR-Cas3 (Type I-C), Cas9 (Type II-A), Cas12 (Cpf1, Type V-A), and Type I restriction-modification (R-M) systems. We propose that ΦKZ utilizes a nucleus-like shell to protect its DNA from attack. Supporting this, we demonstrate that Cas9 is able to cleave ΦKZ DNA in vitro, but not in vivo and that Cas9 is physically occluded from the shell assembled by the phage during infection. Moreover, we demonstrate that the Achilles heel for this phage is the mRNA, as translation occurs outside of the shell, rendering the phage sensitive to the RNA targeting CRISPR-Cas enzyme, Cas13a (C2c2, Type VI-A). Collectively, we propose that the nucleus-like shell assembled by jumbo phages enables potent, broad spectrum evasion of DNA-targeting nucleases.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

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