Abstract:Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoK… Show more
“…It is important to note that, in the absence of ATP hydrolysis, EcoKI is a stable assembly containing all five protein subunits (Dryden et al 1997;Roberts et al 2011). However, for EcoR124I, the binding constants for each of the two HsdR differ by over two orders of magnitude (0.6 nM and 200 nM, respectively), and the two forms of the enzyme, R 1 M 2 S 1 and R 2 M 2 S 1 , are in dynamic equilibrium (Supplemental Material; Supplemental Fig.…”
Section: Resultsmentioning
confidence: 99%
“…As the direction of DNA translocation is defined in the chromatin remodeling translocase, it imposes a similar directionality on each HsdR, and since these have to pull DNA in toward the MTase core of the type I RM enzyme, the orientation of each HsdR relative to the MTase core becomes defined. Assuming that the DNA path between the DNA bound to the HsdR and the DNA bound to the core MTase must not be any longer than ;40 bp, as determined by DNA footprinting experiments (Mernagh et al 1998;Powell et al 1998) and the minimum length of 45 bp of DNA required for ATP hydrolysis (Roberts et al 2011), then the locations of the RecA-like motor domains of the HsdR are forced to be as shown, so that their DNA-binding sites are close to the DNA-binding site of the MTase core. Placement of the HsdR on either side of the MTase and interacting directly with DNA is further supported by the length of the structure of the ArdA anti-restriction DNA mimic protein (Nekrasov et al 2007;McMahon et al 2009), which occupies the entire DNA-binding site on type I RM enzymes.…”
Section: Atomic Modeling Of the Complete Rm Enzymesmentioning
confidence: 99%
“…One may speculate that this great flexibility would allow the RM enzyme to accommodate the stresses built up during the extensive DNA translocation periods observed for these molecular machines. It is noteworthy, in this respect, that a process of deassembly of the enzymes occurs after DNA cleavage, and some of the subunits-although not all and depending on the particular type I RM enzyme-can be reused (Roberts et al 2011;Simons and Szczelkun 2011). Lapkouski et al (2009) proposed a more speculative atomic model of EcoR124I using their structure of HsdR, a postulated DNA path across the subunit, and an early, incomplete model of the MTase core (Obarska et al 2006).…”
Section: Structure Of Type I Restriction Enzymesmentioning
Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.
“…It is important to note that, in the absence of ATP hydrolysis, EcoKI is a stable assembly containing all five protein subunits (Dryden et al 1997;Roberts et al 2011). However, for EcoR124I, the binding constants for each of the two HsdR differ by over two orders of magnitude (0.6 nM and 200 nM, respectively), and the two forms of the enzyme, R 1 M 2 S 1 and R 2 M 2 S 1 , are in dynamic equilibrium (Supplemental Material; Supplemental Fig.…”
Section: Resultsmentioning
confidence: 99%
“…As the direction of DNA translocation is defined in the chromatin remodeling translocase, it imposes a similar directionality on each HsdR, and since these have to pull DNA in toward the MTase core of the type I RM enzyme, the orientation of each HsdR relative to the MTase core becomes defined. Assuming that the DNA path between the DNA bound to the HsdR and the DNA bound to the core MTase must not be any longer than ;40 bp, as determined by DNA footprinting experiments (Mernagh et al 1998;Powell et al 1998) and the minimum length of 45 bp of DNA required for ATP hydrolysis (Roberts et al 2011), then the locations of the RecA-like motor domains of the HsdR are forced to be as shown, so that their DNA-binding sites are close to the DNA-binding site of the MTase core. Placement of the HsdR on either side of the MTase and interacting directly with DNA is further supported by the length of the structure of the ArdA anti-restriction DNA mimic protein (Nekrasov et al 2007;McMahon et al 2009), which occupies the entire DNA-binding site on type I RM enzymes.…”
Section: Atomic Modeling Of the Complete Rm Enzymesmentioning
confidence: 99%
“…One may speculate that this great flexibility would allow the RM enzyme to accommodate the stresses built up during the extensive DNA translocation periods observed for these molecular machines. It is noteworthy, in this respect, that a process of deassembly of the enzymes occurs after DNA cleavage, and some of the subunits-although not all and depending on the particular type I RM enzyme-can be reused (Roberts et al 2011;Simons and Szczelkun 2011). Lapkouski et al (2009) proposed a more speculative atomic model of EcoR124I using their structure of HsdR, a postulated DNA path across the subunit, and an early, incomplete model of the MTase core (Obarska et al 2006).…”
Section: Structure Of Type I Restriction Enzymesmentioning
Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.
“…To circumvent this burden, the organism appears to control the intracellular enzyme concentration. As shown for E. coli, efficient phage restriction occurs by using about 60 molecules of EcoKI, which would consume ϳ0.2% of the total ATP pool (86,87).…”
Section: Fitness Cost Incurred By R-m Systems On Host Bacteriamentioning
SUMMARY
Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.
“…These results contradict those observed by Bianco and Hurley (29). Similar experiments with EcoKI have also found that RecBCD has no effect on turnover of the holoenzyme (45). Figure 5.‘Turnover’ of EcoR124I MTase during DNA cleavage and translocation does not occur and is not influenced by the presence of the exonuclease activity of RecBCD.…”
The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5′-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can ‘turnover’ in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase–nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
