Restriction enzymes methylase

As mentioned previously, bacteria defend themselves from their own restriction enzymes by methylating their DNA. For example, the EcoRI methylase adds a single methyl group to the amino group of... 

Fig. 6. Selection for enzymatic activity (DNA methylation) by compartmentalization. DNA encoding HaeIII methylase is diluted with unrelated DNA (encoding dihydrofolate-reductase). This mixture is dispersed together with a reaction mixture for in vitro transcription and translation to form water in oil compartments. The dilution is chosen such that each compartment contains one DNA molecule on average. In the aqueous compartments, the genes are transcribed and translated. In compartments in which an active methylase is translated, the DNA can be methylated and is subsequently recovered from the emulsion and digested by a restriction enzyme. The methylated DNA (encoding Hae III methylase) is protected against the digestion, remains intact and is subsequently amplified by PCR.

Among the acceptors modified by 5 -adenosylmethionine are specific bases in DNA. The methylation of DNA protects bacterial DNA from cleavage by restriction enzymes (Section 9.3). The base to be methylated is flipped out of the DNA double helix into the active site where it can accept a methyl group from 5 -adenosylmethionine (Figure 24.15). A recurring 5 -adenosylmethionine-binding domain is present in many SAM-dependent methylases. 

A restriction enzyme recognizes and hydrolyzes a particular DNA sequence. The same sequence is recognized and methylated by the partner DNA methylase of the restriction enzyme. A methylated restriction site is immune to cleavage by the restriction enzyme. The methylase keeps the host DNA methylated and thus protected. The invading DNA, if unmethylated itself, vhll be cleaved by the restriction enzyme and subsequently destroyed by less specific nucleases. 

Restriction enzymes require only target DNA, Mg ", and water. DNA methylases require only unmethylated target DNA and S-adenosylmethionine. 

Restriction. The bacterial restriction modification system consists of DNA restriction endonuclease (Pingoud, 2004) and a matched modification enzyme (methylase, i.e. methyltransferase). The restriction endonucleases recognize specific sequences within dsDNA on which the hydrolysis takes place. Three types of restriction enzymes (Table 13.4) have been identified (Yuan, 1981). 

From Boehringer Mannheim, BRL or New England Biolabs all restriction enzymes and EcoRI methylase. 

Another type of R-M system, classified as Type IV, represents an intermediate in the evolutionary pathway between the type II and type III enzymes (4). Type IV enzymes (e.g., Eco57l) consist of separate R-ENase and M-MTase, but the monomeric R ENase possesses an additional methylase activity. The restriction-associated methylase activity is not strong enough to protect the host DNA in... 

Mutation in the regulatory gene (activator/repressor protein) Mutants are resistant to D-cycloserine Structural gene for DNA adenosine methylase. Mutation blocks methylation at the recognition sequences G ATC (methylated on A). Useful in preparing unmethylated DNA which is susceptible to cleavages by such restriction enzymes as Bell and Clal. Dam mutants exhibit lower transformation efficiencies than dam" cells... 

Arrows indicate the phosphodiester bonds cleaved by each restriction endonuclease, Asterisks indicate bases that are methylated by the corresponding methylase (where known), N denotes any base. Note that the name of each enzyme consists of a three-letter abbreviation (in italics) of the bacterial species from which it is derived, sometimes followed by a strain designation and Roman numerals to distinguish different restriction endonucleases isolated from the same bacterial species, Thus BamHI is the first (I) restriction endonuclease characterized from Bacillus amyloliquefaciens, strain H. 

Type II restriction-modification systems differ from their type I and type III counterparts in that the endonuclease and DNA methylase activities are conducted by two separate enzymes (not a single multisubunit complex). The restriction endonuclease cleaves both strands of the DNA duplex within a defined recognition sequence, while the companion DNA methylase methylates a specific base within the same recognition sequence. In contrast... 

Roberts, R. J., and D. Macelis. 1997. REBASE—restriction en-Z3unes and methylases. Nucl. Acids Res. 25 248-262. Information on accessing a continuously updated database on restriction and modification enzymes at http //www.neb.com/rebase. 

In prokaryotic DNA the major methylated bases are N -methyladenine (mA) and to a lesser extent Nl-methylcytosine. Methylation in bacteria occurs at specific sites. In E. coli, methylation of A residues in the sequence 5 -GATC-3 is involved in mismatch error correction, and it plays a role in controlling initiation of DNA replication. Methylation at other sites protects DNA against cleavage by restriction endonucleases (described here). Structural studies on a bacterial DNA methylase have shown that the bases undergoing methylation rotate completely out of the DNA duplex and into a catalytic pocket within the enzyme structure. Other enzymes that work on bases, such as uracil-N-glycosylase, operate similarly. 

Restriction-modification is a term for bacterial enzyme systems that cleave DNA sequences. Each system consists of two distinct enzyme activities a DNA methylase and an endonuclease that catalyzes the double-strand DNA break. Type I restriction endonuclease systems have both methylase and nuclease activities in one protein molecule, which contains three subunits. One subunit contains the nuclease, one the methylase, and one a sequence recognition determinant. The recognition site is not symmetrical, and cleavage occurs some distance (up to 10 kbp) away from the recognition site, although methylation occurs within the recognition site. 

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