The enzymology of in vitro DNA recombination

Abstract

The ease with which DNA molecules can now be joined in vitro is a consequence of the availability of restriction endonucleases, enzymes which recognise specific sequences in DNA and then cleave both strands of the duplex. These enzymes have been found in many prokaryotes and are likely to be responsible for the degradation of ‘alien’ DNA molecules, the indigenous DNA being protected from degradation by a modification enzyme, usually a methylase. Restriction endonucleases are responsible for the phenomenon of host controlled modification of bacteriophage, first described in the early 50s [reviewed in Ref. 1]. If phage λ, which has been propagated on E. coli strain K, is then allowed to infect E. coli strain B, the efficiency of the infectious process is very low. The phage produced from this infection can, however, reinfect E. coli strain B with high efficiency. Three genetic loci can be identified which control this system: hsdS, hsdM and hsdR. A polypeptide which governs the specificity of the system is determined by hsdS. The gene product of hsdM is the modification enzyme which also interacts with the product of the hsdR gene, the restriction endonuclease, in the cleavage process. In the above examples the phage grown on strain K would have been modified at sites specified by the K restriction-modification system. In the first infective cycle in E. coli B cells the B restriction-modification system detects the absence of B modification and degrades the infecting DNA. A small proportion of molecules are, however, methylated by the B modification system, and these survive restriction on the next infective cycle. This phenomenon has to be borne in mind when introducing foreign unmodified in vitro recombinant DNA into E. coli. In order for these molecules to survive the recipient strain should have defective hsdS or hsdR genes.