Host-controlled Restriction and Modification

Host-controlled Restriction and Modification
Host-controlled restriction and modification arc most readily observed when bacteriophages are transferred from one bacterial host strain to another. If a stock preparation of phage X, for example, is made by growth

upon E. coil strain C and this stock is then titred upon E. coil C and E. coil K, the titres observed on these two strains will differ by several orders of magnitude, the litre on E. coil K being he lower. The phage are said to be restricted by the second host strain (E. coil K). When those phage that do result from the infection of E. coil K are now replated on E. coil K they are no longer restricted; but if they are first cycled through E. coil C they are once again restricted when plated upon E. coil K (Fig. 2.1). Thus the efficiency with which phage plates upon a particular host strain depends upon the strain on which it was last propagated. This non-heritable change conferred upon the phage by the second host strain (E. coil K) that allows it to be replated on that strain without further restriction is called modification. The restricted phages adsorb to restrictive hosts and inject their DNA normally. When the phage are labelled with 32P it is apparent that their
DNA is degraded soon after injection (Dussoix & Arbcr 1962) and the
      endonuclcase that is primarily responsible for this degradation is called a resirkilon endonuclease or restriction enzyme. The restrictive host must of course protect its own DNA from the potentially lethal effects of the restriction endonuclcase and so its DNA must be appropriately modified. Modification involves meihylation of certain bases at a very limited number of sequences within DNA which constitute the recognition sequences for the restriction endonuclease. This explains why phage that survive one cycle of
growth upon the restrictive host can subsequently reinfect that host efficiently; their DNA has been replicated in the presence of the modifying mechylase and so it, like the host DNA, becomes methylated and protected
from the restriction system. Although phage infection has been chosen as our example to illustrate restriction and modification, these processes can occur whenever DNA is transferred from one bacterial strain to another. Conjugation, transduction, transformation and transfcction arc all subject to the constraint of host-
controlled restriction. The genes that specify host-controlled restriction and modification systems may reside upon the host chromosome itself or may be located on a plasmid or prophage such as P1.

       The restriction endonucicase of E. cob K was the first to be isolated and studied in detail. Meselson & Yuan l968) achiesed this by devising an sngcrnous assay in which a fractionated cell extract was incubated with a mixture of unmodified and modified phage ). DNAs which werc differentially radiolabeLled—one with 3H, the oilier with 11P—so that they could be distinguished. After incubation, the DNA mixture was analysed by sedimentation through a sucrose gradient where the appearance of degraded unmodified DNA in the presence of undegraded modified DNA indicated the activity of restriction endonuclcase. The enzyme from E. cvii K, and the simib, one from E. rob H, were found to have unusual properties. In addition to magnesium ions, they require the cofactoes ATP and S-adenosyl-mcthionine, and DNA degradation
in vitro is accompanied by hydrolysis of the ATP in amounts greatly exceeding the stoichiomeiry of DNA breakage (Bickle et a!. 1978). In addition, the enzymes are now known to interact with an unmodified
recognition sequence in duplex DNA and then surprisingly, to track along the DNA molecule. The enzyme from E. cvii II is known to track to one sak only of the recognition sequence, which is asymmetric Alter traselling for a distance corresponding to between 1000 and $000 nuclcotidcs it clcascs one strand only of the DNA at an apparently random site, and makes a gap about 75 nucleotides in length by releasing acid-soluble oligonucleorides. There is no evidence that the enzyme is truly catalytic, and having acted
once in this way, a second enzyme molecule is required to complete the double-strand break (Rosamond et vi. 1979). Enzymes with these properties are now known as type I restriction endonucicases. Their biochemistry still presents many puzzles. For instance, the precise role of S.adcnosylm ethionine remains unclear.

      While these bizarre properties of type I restriction enzymes were being unravelled, a rcstriction endonuclcasc from Huemophilus influenzae Rd was discovered (Kelly & Smith 1970, Smith & Wilcox 1970) thai was to become the prototype of a large number of resiriction endonucleases— now known as type II enzymes—that have none of the unusual properties displayed by type I enzymes and which arc fundamentally important in thc manipulation of DNA. The type II enzymes recognize a particular target
sequence in a duplex DNA molecule and break the polynucleotide chains within that sequence to give rise to discrete DNA fragments of dcfincd length and sequence. In Fact, the activity of these enzymes is often assayed and studied by gel electrophoresis of the DNA fragments which they generate (see Fig. 1.2). As expected, digests of small plasmid or viral DNAs give characteristic simple DNA band patterns.
     Very many type II restriction cndonucleascs have now been isolated from a wide variety of bacteria. In a recent review, Roberts (1978) lists 168 enzymes that have been at least partially characterized, and (he number continues to grow as more bacterial genera are surveyed for their presence. Ii is worth noting that many so-called restriction endonucleases have not formally been shown to correspond with any genetically identified restriction and modification system of the bacteria from which they have been prepared: it is usually assumed that a site-specific cndonuctcasc which is  inactive upon hosi DNA and active upon exogenous DNA is, in fact, a restriction cndonuclcase.

• nomencleture
• Machanical Sharing of DNA
• Joining DNA Molecules