Highlights of the DNA cutters: a short history of the restriction enzymes.

In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

A third family of allelic hsd genes in Salmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA.

Salmonella enterica serovar blegdam has a restriction and modification system encoded by genes linked to serB. We have cloned these genes, putative alleles of the hsd locus of Escherichia coli K-12, and confirmed by the sequence similarities of flanking DNA that the hsd genes of S. enterica serovar blegdam have the same chromosomal location as those of E. coli K-12 and Salmonella enterica serovar typhimurium LT2. There is, however, no obvious similarity in their nucleotide sequences, and while the gene order in S. enterica serovar blegdam is serB hsdM, S and R, that in E. coli K-12 and S. enterica serovar typhimurium LT2 is serB hsdR, M and S. The hsd genes of S. enterica serovar blegdam identify a third family of serB-linked hsd genes (type ID). The polypeptide sequence predicted from the three hsd genes show some similarities (18-50% identity) with the polypeptides of known and putative type I restriction and modification systems; the highest levels of identity are with sequences of Haemophilus influenzae Rd. The HsdM polypeptide has the motifs characteristic of adenine methyltransferases. Comparisons of the HsdR sequence with those for three other families of type I systems and three putative HsdR polypeptides identify two highly conserved regions in addition to the seven proposed DEAD-box motifs.

The impact of phage lambda: from restriction to recombineering.

Experiments using phage lambda provided early insights into important molecular mechanisms, including genetic recombination and the control of gene expression. Before recombinant DNA technology, the use of lambda, most particularly lambda transducing phages, illustrated the importance of cloning bacterial genes, already providing some insight into how to use cloned genes to advantage. Subsequently, lambda made significant contributions to recombinant DNA technology, including the early generation of genomic and cDNA libraries. More recently, lambda genes associated with recombination have enabled techniques referred to as 'recombineering' to be developed. These techniques permit the refined manipulation, including mutation, of foreign genes in Escherichia coli and their subsequent return to the donor organism.

Control of the endonuclease activity of type I restriction-modification systems is required to maintain chromosome integrity following homologous recombination.

A type I restriction-modification enzyme will bind to an unmethylated target sequence in DNA and, while still bound to the target, translocate DNA through the protein complex in both directions. DNA breakage occurs when two translocating complexes collide. However, if type I restriction-modification systems bind to unmodified target sequences within the resident bacterial chromosome, as opposed to incoming 'foreign' DNA, their activity is curtailed; a process known as restriction alleviation (RA). We have identified two genes in Escherichia coli, rnhA and recG, mutations in which lead to the alleviation of restriction. Induction of RA in response to these mutations is consistent with the production of unmodified target sequences following DNA synthesis associated with both homologous recombination and R-loop formation. This implies that a normal function of RA is to protect the bacterial chromosome when recombination generates unmodified products. For EcoKI, our experiments demonstrate the contribution of two pathways that serve to protect unmodified DNA in the bacterial chromosome: the primary pathway in which ClpXP degrades the restriction endonuclease and a mechanism dependent on the lar gene within Rac, a resident, defective prophage of E. coli K-12. Previously, the potential of the second pathway has only been demonstrated when expression of lar has been elevated. Our data identify the effect of lar from the repressed prophage.

Is modification sufficient to protect a bacterial chromosome from a resident restriction endonuclease?

It has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system. But, when target sequences within the chromosome of one such bacterium (Escherichia coli K-12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated. Thus, the resident chromosome is recognized as 'self' rather than 'foreign' even in the absence of modification. We now provide evidence that restriction alleviation may be a characteristic of Type I restriction-modification systems, and that it can be achieved by different mechanisms. Our experiments support disassembly of active endonuclease complexes as a potential mechanism. We identify amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrate that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment. In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome.