The other face of restriction: modification-dependent enzymes.

The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980's, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction). The new class of modification-dependent restriction enzymes was named Type IV, as distinct from the familiar modification-blocked Types I-III. A third Escherichia coli enzyme, mrr (modified DNA rejection and restriction) recognizes both methylcytosine and methyladenine. In recent years, the universe of modification-dependent enzymes has expanded greatly. Technical advances allow use of Type IV enzymes to study epigenetic mechanisms in mammals and plants. Type IV enzymes recognize modified DNA with low sequence selectivity and have emerged many times independently during evolution. Here, we review biochemical and structural data on these proteins, the resurgent interest in Type IV enzymes as tools for epigenetic research and the evolutionary pressures on these systems.

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.

No threshold for the induction of chromosomal damage at clinically relevant low doses of X rays.

The recent steep increase in population dose from radiation-based medical diagnostics, such as computed tomography (CT) scans, requires insight into human health risks, especially in terms of cancer development. Since the induction of genetic damage is considered a prominent cause underlying the carcinogenic potential of ionizing radiation, we quantified the induction of micronuclei and loss of heterozygosity events in human cells after exposure to clinically relevant low doses of X rays. A linear dose-response relationship for induction of micronuclei was observed in human fibroblasts with significantly increased frequencies at doses as low as 20 mGy. Strikingly, cells exposed during S-phase displayed the highest induction, whereas non S-phase cells showed no significant induction below 100 mGy. Similarly, the induction of loss of heterozygosity in human lymphoblastoid cells quantified at HLA loci, was linear with dose and reached significance at 50 mGy. Together the findings favor a linear-no-threshold model for genetic damage induced by acute exposure to ionizing radiation. We speculate that the higher radiosensitivity of S-phase cells might relate to the excessive cancer risk observed in highly proliferative tissues in radiation exposed organisms.

Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra.

Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4 PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C>T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

Tracking EcoKI and DNA fifty years on: a golden story full of surprises.

1953 was a historical year for biology, as it marked the birth of the DNA helix, but also a report by Bertani and Weigle on 'a barrier to infection' of bacteriophage lambda in its natural host, Escherichia coli K-12, that could be lifted by 'host-controlled variation' of the virus. This paper lay dormant till Nobel laureate Arber and PhD student Dussoix showed that the lambda DNA was rejected and degraded upon infection of different bacterial hosts, unless it carried host-specific modification of that DNA, thus laying the foundations for the phenomenon of restriction and modification (R-M). The restriction enzyme of E.coli K-12, EcoKI, was purified in 1968 and required S-adenosylmethionine (AdoMet) and ATP as cofactors. By the end of the decade there was substantial evidence for a chromosomal locus hsdK with three genes encoding restriction (R), modification (M) and specificity (S) subunits that assembled into a large complex of >400 kDa. The 1970s brought the message that EcoKI cut away from its DNA recognition target, to which site the enzyme remained bound while translocating the DNA past itself, with concomitant ATP hydrolysis and subsequent double-strand nicks. This translocation event created clearly visible DNA loops in the electron microscope. EcoKI became the archetypal Type I R-M enzyme with curious DNA translocating properties reminiscent of helicases, recognizing the bipartite asymmetric site AAC(N6)GTGC. Cloning of the hsdK locus in 1976 facilitated molecular understanding of this sophisticated R-M complex and in an elegant 'pas de deux' Murray and Dryden constructed the present model based on a large body of experimental data plus bioinformatics. This review celebrates the golden anniversary of EcoKI and ends with the exciting progress on the vital issue of restriction alleviation after DNA damage, also first reported in 1953, which involves intricate control of R subunit activity by the bacterial proteasome ClpXP, important results that will keep scientists on the EcoKI track for another 50 years to come.