Abstract:Background: Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric, complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a pr… Show more
“…Cleavage results when McrC associates with full-length McrB:GTP complex bound to DNA and GTP is hydrolyzed (72). LlaJI, a modification-blocked restriction activity, exhibits a similar organization (85), although cleavage could not be demonstrated in vitro . Figure 3.McrBC Assembly Model.…”
Section: Genetics Biochemistry and Structuresmentioning
The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980’s, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction). The new class of modification-dependent restriction enzymes was named Type IV, as distinct from the familiar modification-blocked Types I–III. A third Escherichia coli enzyme, mrr (modified DNA rejection and restriction) recognizes both methylcytosine and methyladenine. In recent years, the universe of modification-dependent enzymes has expanded greatly. Technical advances allow use of Type IV enzymes to study epigenetic mechanisms in mammals and plants. Type IV enzymes recognize modified DNA with low sequence selectivity and have emerged many times independently during evolution. Here, we review biochemical and structural data on these proteins, the resurgent interest in Type IV enzymes as tools for epigenetic research and the evolutionary pressures on these systems.
“…Cleavage results when McrC associates with full-length McrB:GTP complex bound to DNA and GTP is hydrolyzed (72). LlaJI, a modification-blocked restriction activity, exhibits a similar organization (85), although cleavage could not be demonstrated in vitro . Figure 3.McrBC Assembly Model.…”
Section: Genetics Biochemistry and Structuresmentioning
The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980’s, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction). The new class of modification-dependent restriction enzymes was named Type IV, as distinct from the familiar modification-blocked Types I–III. A third Escherichia coli enzyme, mrr (modified DNA rejection and restriction) recognizes both methylcytosine and methyladenine. In recent years, the universe of modification-dependent enzymes has expanded greatly. Technical advances allow use of Type IV enzymes to study epigenetic mechanisms in mammals and plants. Type IV enzymes recognize modified DNA with low sequence selectivity and have emerged many times independently during evolution. Here, we review biochemical and structural data on these proteins, the resurgent interest in Type IV enzymes as tools for epigenetic research and the evolutionary pressures on these systems.
“…Type II DNA MTases are enzymes found ubiquitously in the prokaryotic world and play important additional roles (other than in host protection from invading DNA) in several cellular processes, such as replication, transcription, and population evolution (13). Type II methyltransferases function by transferring a methyl group from S-adenosyl-L-methionine (SAM) to a target sequence, and these MTases can be divided into three functional classes.…”
“…Although previous studies show LlaJI.R1 binds DNA sitespecifically (29,31), the molecular means through which this is achieved remains unknown. Numerous attempts to purify either the full-length L. lactis LlaJI.R1 or its isolated N-terminal DNA-binding domain for structural studies were unsuccessful.…”
Section: Structure and Topology Of The Hpllajir1 N-terminal Domainmentioning
confidence: 97%
“…The LlaJI restriction cassette was first identified in Lactococcus lactis on the naturally occurring plasmid pNP40 and shown to confer resistance against common lactococcal phages (28). It consists of an operon encoding two 5mC-methyltransferases, LlaJI.M1 and LlaJI.M2, and two restriction proteins, LlaJI.R1 and LlaJI.R2, both of which are absolutely required for restriction activity in vivo (29). The M1 and M2 methyltransferase activities modulate expression of LlaJI operon in vivo (30).…”
Section: This Work Was Supported By Cornell University and National Imentioning
confidence: 99%
“…Unlike McrB, however, L. lactis LlaJI.R1 binds DNA sitespecifically, recognizing the asymmetric 5Ј-GACGC-3Ј sequence in one strand and 5Ј-GCGTC-3Ј in the other strand (29). Other LlaJI homologs have been identified in Helicobacter pylori, Streptococcus pyogens, Bacillus cereus, and Clostridium cellulovorans (29,31). Of these, C. cellulovorans LlaJI has also been shown to target the same asymmetric, 5-bp sequence (31).…”
Section: This Work Was Supported By Cornell University and National Imentioning
Restriction modification systems consist of an endonuclease that cleaves foreign DNA site-specifically and an associated methyltransferase that protects the corresponding target site in the host genome. Modification-dependent restriction systems, in contrast, specifically recognize and cleave methylated and/or glucosylated DNA. The LlaJI restriction system contains two 5-methylcytosine (5mC) methyltransferases (LlaJI.M1 and LlaJI.M2) and two restriction proteins (LlaJI.R1 and LlaJI.R2). LlaJI.R1 and LlaJI.R2 are homologs of McrB and McrC, respectively, which in function together as a modification-dependent restriction complex specific for 5mC-containing DNA. LlaJI.R1 binds DNA site-specifically, suggesting that the LlaJI system uses a different mode of substrate recognition. Here we present the structure of the N-terminal DNA-binding domain of LlaJI.R1 at 1.97-Å resolution, which adopts a B3 domain fold. Structural comparison to B3 domains in plant transcription factors and other restriction enzymes identifies key recognition motifs responsible for site-specific DNA binding. Moreover, biochemistry and structural modeling provide a rationale for how LlaJI.R1 may bind a target site that differs from the 5-bp sequence recognized by other LlaJI homologs and identify residues critical for this recognition activity. These findings underscore the inherent structural plasticity of B3 domains, allowing recognition of a variety of substrates using the same structural core.
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