Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease

The BcgI endonuclease exemplifies a subset of restriction enzymes, the Type IIB class, which make two double-strand breaks (DSBs) at each copy of their recognition sequence, one either side of the site, to excise the sequence from the remainder of the DNA. In this study, we show that BcgI is essentially inactive when bound to a single site and that to cleave a DNA with one copy of its recognition sequence, it has to act in trans, bridging two separate DNA molecules. We also show that BcgI makes the two DSBs at an individual site in a highly concerted manner. Intermediates cut on one side of the site do not accumulate during the course of the reaction: instead, the DNA is converted straight to the final products cut on both sides. On DNA with two sites, BcgI bridges the sites in cis and then generally proceeds to cut both strands on both sides of both sites without leaving the DNA. The BcgI restriction enzyme can thus excise two DNA segments together, by cleaving eight phosphodiester bonds within a single-DNA binding event

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

<p>Abstract</p> <p>Background</p> <p>The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.</p> <p>Results</p> <p>Fusion of <it>eco29kIR </it>and <it>M </it>ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. <it>In vitro </it>the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. <it>In vivo </it>new RMS can provide a 10²-fold less protection of host cells against phage invasion.</p> <p>Conclusions</p> <p>We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.</p

Type I restriction enzymes and their relatives

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes

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

TstI, a Type II restriction–modification protein with DNA recognition, cleavage and methylation functions in a single polypeptide

Type II restriction–modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conven-tional Type II schemes as they employ the same pro-tein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study anal-ysed one such single-chain protein, TstI. Compar-ison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the het-erologous assembly of BcgI, TstI forms a homote-tramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites