Complex restriction enzymes: NTP-driven molecular motors
Introduction
Natural selection is one of the primary forces in nature and bacterial survival depends heavily on the cells’ ability to protect themselves against bacteriophage attacks/infections. One of the mechanisms that bacteria have evolved for this purpose is nucleoside triphosphate-dependent (NTP) restriction enzymes (for previously published reviews, see [1], [2], [3], [4], [5]). Restriction–modification (R–M) enzymes have the dual opposing functions of: (i) protecting the host DNA against restriction by methylating the DNA within specific target sites, and (ii) “restricting”, i.e. degrading any unmodified DNA that may enter the cell. Based on their particular subunit structures and co-factor requirement, R–M enzymes have been classified into three main groups. Type II R–M enzymes comprise separate endonucleases and methylases that act independently from each other and have as a sole common factor the ability to target for cleavage or methylation, respectively, a single specific DNA sequence. This recognition site is typically a 4–8 bp palindromic sequence. Type II restriction enzymes have simple co-factor requirements: restriction depends on the presence of Mg2+, and modification requires S-adenosyl-methionine (AdoMet). Type I R–M systems are hetero-oligomeric enzymes constituted of three subunits: HsdR (host specificity for DNA, restriction), HsdM (modification or methylation) and HsdS (specificity) encoded by the hsd genes (hsdR, hsdM and hsdS, respectively) encoded contiguously on the bacterial genome. Type I restriction enzymes are present in two different oligomeric forms in vivo. The M2S form containing two HsdM and one HsdS subunits catalyses methylation of the DNA in the presence of the co-factors AdoMet and Mg2+ [6], [7], [8], [9], whereas the multimer R2M2S (M2S + two HsdR subunits) requires the presence of three co-factors, AdoMet, Mg2+ and ATP, for restriction of unmethylated DNA. Although it was originally thought that type I restriction enzymes might be restricted to the Enterobacteriaceae [1], [3], the genome sequencing projects developed in the last few years have revealed their presence in a large number of bacterial genera. A list of all type I restriction enzymes identified so far has been compiled and is maintained on the REBASE web site [10]. Type III R–M enzymes are composed of two subunits, products of the mod and res genes [11], [12]. The Mod subunit forms a stable dimer that acts as an independent modification methylase in the presence of AdoMet. In contrast, the Res subunit has no enzymatic activity when not complexed with Mod. The complex Res2Mod2 mediates DNA cleavage in the presence of ATP and the reaction is stimulated by AdoMet. This bacterial defence mechanism array is complemented by modification-dependent restriction (MDR) systems that specifically recognise and cut modified DNA. These MDR endonucleases have no associated methylase. The best-characterised member of MDR enzymes, McrBC, is a two-subunit complex that has a GTP-hydrolysis activity and a strict requirement for methylated DNA as a substrate. This review concentrates on the ATP-dependent type I and type III restriction–modification enzymes, on the GTP-hydrolysing McrBC complex and their respective functions.
Section snippets
Type I R–M enzymes
Type I R–M enzymes are multi-functional complexes. They catalyse many different functions such as recognition of and binding to a specific DNA sequence, methylation of specific adenine residues within that same sequence, DNA translocation coupled to ATP hydrolysis and, finally, DNA cleavage. Although the subunits of type I R–M enzymes cannot act independently from the complex, each of the functions enumerated above can be attributed specifically to one of the three subunits. The recognition
Type III R–M enzymes
Type III restriction–modification enzymes are hetero-oligomeric, multi-functional enzymes composed of the Mod subunit responsible for site-specific methylation of the DNA and the Res subunit that is able, after formation of a (Mod)2(Res)2 complex, to translocate and cleave DNA in an ATP-dependent fashion. The recognition sequence of type III enzymes is asymmetric, uninterrupted and 5–6 bp in length. Cleavage requires the presence of inversely oriented recognition sites and occurs at a distance
Modification-dependent restriction enzymes
Historically, modification-dependent restriction (MDR) endonucleases represent the first restriction systems ever described [86], predating even the well-known, classical phage assay experiments that led to the identification of E. coli type I R–M systems [87], [88]. The first studied MDR enzymes were aimed at T-even phages. They exhibited restriction activity against non-glycosylated 5-hydroxymethylcytosine (HMC)-DNA in T-even phages, and were therefore named Rgl for restricts glucose-less
Conclusion
In this review, we have described three bacterial restriction systems that belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5′-triphosphate to translocate DNA and thus trigger DNA cleavage randomly, away from the recognition site. Of the three different restriction systems described here, two were restriction–modification systems and utilised ATP, i.e. type I and type III enzymes, and one, the modification-dependent endonuclease, McrBC,
Acknowledgements
Work from this laboratory was supported by the Swiss National Science Foundation.
References (116)
- et al.
EcoA: the first member of a new family of type I restriction modification systems. Gene organization and enzymatic activities
J. Mol. Biol.
(1985) - et al.
High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124
Gene
(1992) - et al.
Purification and characterization of the methyltransferase from the type 1 restriction and modification system of Escherichia coli K12
J. Biol. Chem
(1993) - et al.
DNA restriction–modification genes of phage P1 and plasmid p15B. Structure and in vitro transcription
J. Mol. Biol
(1983) - et al.
Type III DNA restriction and modification systems EcoP1 and EcoP15. Nucleotide sequence of the EcoP1 operon, the EcoP15 mod gene and some EcoP1 mod mutants
J. Mol. Biol
(1988) - et al.
Conservation of complex DNA recognition domains between families of restriction enzymes
Cell
(1989) - et al.
Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes
J. Mol. Biol
(1989) - et al.
Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences
J. Mol. Biol
(1985) - et al.
Sequence diversity among related genes for recognition of specific targets in DNA molecules
J. Mol. Biol
(1983) - et al.
Surface labelling of the type I methyltransferase M.EcoR124I reveals lysine residues critical for DNA binding
J. Mol. Biol
(1996)
A novel mutant of the type I restriction–modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway
J. Mol. Biol
Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes
J. Mol. Biol
Mutations that confer de novo activity upon a maintenance methyltransferase
J. Mol. Biol
DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine
J. Mol. Biol
The DNA binding characteristics of the trimeric EcoKI methyltransferase and its partially assembled dimeric form determined by fluorescence polarisation and DNA footprinting
J. Mol. Biol
Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme
J. Mol. Biol
Endonuclease (R) subunits of type-I and type-III restriction- modification enzymes contain a helicase-like domain
FEBS Lett
On the structure and operation of type I DNA restriction enzymes
J. Mol. Biol
Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism
Cell
The DNA translocation and ATPase activities of restriction-deficient mutants of EcoKI
J. Mol. Biol
The deoxyribonucleic acid modification and restriction enzymes of Escherichia coli B. II. Purification, subunit structure, and catalytic properties of the restriction endonuclease
J. Biol. Chem.
DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI
J. Mol. Biol
The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties
J. Mol. Biol
DNA cleavage by the type IC restriction–modification enzyme EcoR124II
J. Mol. Biol
Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy
Biophys. J.
ATPase activityof the type IC restriction–modification system EcoR124II
J. Mol. Biol
Functional analysis of conserved motifs in EcoP15I DNA methyltransferase
J. Mol. Biol
Interaction of EcoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5'-CAGCAG-3'
J. Mol. Biol.
DNA recognition by the EcoP15I and EcoPI modification methyltransferases
Gene
Probing the role of cysteine residues in the EcoP15I DNA methyltransferase
J. Biol. Chem
Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence
J. Mol. Biol.
S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes
J. Mol. Biol
Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I
J. Mol. Biol
DNA cleavage by type III restriction–modification enzyme EcoP15I is independent of spacer distance between two head to head oriented recognition sites
J. Mol. Biol
Host specificity of DNA produced by Escherichia coli. I. Host controlled modifiation of bacteriophage lambda
J. Mol. Biol
Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains
Gene
Biology of DNA restriction
Microbiol. Rev
The ATP-dependent restriction enzymes
DNA restriction and modification systems
Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle)
Microbiol. Mol. Biol. Rev.
Nucleoside triphosphate-dependent restriction enzymes
Nucleic Acids Res
Purification and biochemical characterisation of the EcoR124 type I modification methylase
Nucleic Acids Res
REBASE – restriction enzymes and methylases
Nucleic Acids Res
ATP-induced conformational changes in the restriction endonuclease from Escherichia coli K-12
Proc. Natl. Acad. Sci. USA
Translocation and specific cleavage of bacteriophage T7 DNA in vivo by EcoKI
Proc. Natl. Acad. Sci. USA
Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement
EMBO J
Reassortment of DNA recognition domains and the evolution of new specificities
Mol. Microbiol
A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes
Nucleic Acids Res
Localization of a protein–DNA interface by random mutagenesis
EMBO J
The specificity of StySKI, a type I restriction enzyme, implies a structure with rotational symmetry
Nucleic Acids Res
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