Type I restriction endonuclease

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. 

The vast majority of biochemical reactions are catalyzed by enzymes (Bagg, 2004 Copeland, 2000), which are proteins fulfilling all the three criteria of being biocatalysts efficiency (a), specificity (b) and multiple turnover (c). The term enzymes is reserved for the natural catalytic proteins in this text. Although few quasi-catalytic enzymes are known such as type I restriction endonuclease, poly(ADP-ribose) synthetase and transmethylase for 0 -methylguanine mediate specific reactions with large acceleration but are inactivated in the reaction. 

There are three types of restriction endonucleases, designated I, II, and III. Types I and HI are generally large, multisubunit complexes containing both the endonucle-... 

The type II restriction endonucleases, which are the ones most widely used in molecular biology, are relatively small 50- to 100-kDa monomeric or dimeric proteins. About 2400 different enzymes with 188 different specificities had been isolated by 1995.83/84 The sites of attack, in most instances, are nucleotide sequences with a twofold axis of local symmetry.85 For example, the following sites of cleavage have been identified for two restriction endonucleases encoded by the DNA of R-factor plasmids of E. coli and for a restriction enzyme from Hemophilus influenzae. In the diagrams i are sites of cleavage, are sites of methyla-tion, and are local twofold axes (centers of palindromes) N can be any nucleotide with a proper base pairing partner. 

Fig. 13.2. Cleavage of the specific recognition sites by the type II restriction endonucleases EcoRV, EcoRI and Bg/I The cleavage reaction, which requires Mg2+ as cofactor, leads to 5 phosphate and 3 OH ends. While EcoRV cleavage results in blunt ends, EcoRI and Bg/I generate sticky ends with a 5 and 3 overhang, respectively.

Alu repeats are approximately 300 base pairs in length. They got their name from the foot that most carry within them the base sequence AGCT the recognition site for the Alu I restriction endonuclease, a type of enzyme that cuts DNA at a specific site. There are over 500,000 Alu repeats scattered throughout the human genome. On average, one can be found every 4,000 base pairs along a human DNA molecule. How they arose is still a matter... 

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... 

The MAPDRAW application provides a detailed restriction map of the DNA sequence of interest, whether it has been entered manually or imported from a database. Since MAPDRAW is able to identify 478 different restriction endonuclease recognition sequences, you may wish to simplify your restriction map by applying selective filters. These filters will identify restriction endonuclease sites specifically on the basis of name, the 5 or 3 single-stranded overhangs that they produce, the frequency (number) of sites contained within the sequence, and/or the restriction endonuclease class (type I or type II, see Section V). In addition, a detailed de-... 

These enzymes are extraordinarily abundant over 1200 restriction endonucleases had been isolated and characterized by early 1990. Of three classes defined, type II restriction enzymes, which generally cut within their recognition sequences, have found uses in a host of biomedical research and diagnostic applications to be discussed below. Type 1 enzymes cut nonspecifically many nucleotides distal to specific recognition sequences and contain both restriction enzyme and DNA modification (see below) activities on different subunits of multienzyme complexes. Type III restriction enzymes share the multienzyme aspeas of type I enzymes but vary in other properties such as ATPase activity and cofactor requirements. 

The archetypal type I system is that of E.coli K12, EcoKl. This enzyme comprises three different subunits, the specificity (S) subunit which recognises the DNA sequence 5 AAC-(N)6-GTGC3 , the modification mtase (M) subunit, and the restriction endonuclease (R) subunit. Two M subunits bind to one S subimit to form an active modification mtase which... 

The role of the divalent metal ions present in natural phosphodiesterases became clear in bovine pancreatic deoxyribonuclease I (DNase I), the first endonuclease structure determined by X-ray crystallography. The nucleophilic attack of a water molecule activated by a histidine residue is facilitated by the interaction of a calcium ion with the phosphate group to be cleaved (291). Glutamic and aspartic residues involved in magnesium binding have been identified in the crystal structure of four type II restriction enzymes EcoRl (292), EcoRV (293), Pvull (294), and BamHl (295), as well as in that of the repair... 

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). 

Restriction endonucleases are produced by many bacteria and hundreds of different types of these en mes have been purified from different organisms. These entymes have a property to cut double stranded DNA in a sequence specific manner, i.e., any given restriction enzyme will cut the DNA at one and only one base sequence. An example is given below. ... 

Plasmid DNA is an independent type of DNA, which occurs naturally in many microorganisms in addition to the genomic DNA of the respective organism. Plasmids are comparatively small (typically 5-10 kb ), circular DNA molecules that can multiply independendy from the genomic (chromosomal) DNA. They occur naturally in the supercoiled (major percentage) and the open circular form (see Fig. 2.2). Linearized fragments of plasmid DNA can be obtained by digestion of the plasmid with restriction endonucleases, i.e., enzymes that cut the DNA at specific base pair sequences. ... 

Type II restriction enzymes (R ENases) are a large group of DNA endonucleases that are distinguished from other R—M systems by two characteristics (i) the ENase activity for DNA restriction is physically and functionally separate from... 

---