On the DNA cleavage mechanism of Type I restriction enzymes.

Although the DNA cleavage mechanism of Type I restriction-modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5'- and 3'-overhangs of varying lengths. EcoAI preferentially generated 3'-overhangs of 2-3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5'-overhangs of a length of approximately 6-7 and 3-5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.

Restricting restriction.

Systems biology is a new, fashionable and well-funded discipline, which to quote from a recent review aims to 'examine the structure and dynamics of cellular and organismal function, rather than the characteristics of isolated parts of a cell or organism em leader ' (Kitano, H. (2002) Science 295:1662-1664). Systems biology will do this by profiting from the vast amounts of biological information that are available in the genomics era and make extensive use of computer modelling. But: 'many breakthroughs in experimental devices, advanced software and analytical methods are required before the achievements of system biology can live up to their much-touted potential'. This edition of Molecular Microbiology contains a paper that is the product of traditional experimental biology but which could serve as a test case for systems biology. The paper shows how bacteria integrate such disparate subsystems as DNA restriction, homologous recombination and regulated proteolysis to protect their chromosomes from degradation. When systems biology can predict this level of choreography, it will be a mature discipline.

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme.

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.

Chemical restriction: strand cleavage by ammonia treatment at 8-oxoguanine yields biologically active DNA.

Cleavage of DNA single and double strands at an 8-oxoguanine-containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH(3) and O(2) at 60 degrees C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single-stranded overhangs on PCR-amplified DNA that can be ligated. As an example, the lac Z' gene was amplified by PCR with 8-oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3'-overhang independent of the sequence of the cloned DNA.

Characterization and mutational analysis of the RecQ core of the bloom syndrome protein.

Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the Escherichia coli DNA helicase RecQ, a suppressor of illegitimate recombination. After over-production in E.coli, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642-1290). The BLM(642-1290) fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM(642-1290) exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg(2+) and ATPgammaS. Rates of ATP hydrolysis and DNA unwinding by BLM(642-1290) showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a lambda Spi(-) assay, we have found that the BLM(642-1290) fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in E.coli. A deletion of 182 C-terminal amino acid residues of BLM(642-1290), including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM(642-1290) values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.

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.

Lack of regulation of the modification-dependent restriction enzyme McrBC in Escherichia coli.

Restriction alleviation (RA) by the type I restriction enzyme EcoKI is caused by treatments that damage DNA. RA is due to proteolysis of the EcoKI HsdR subunit by the ClpXP ATP-dependent protease. Here we show that the modification-dependent enzyme McrBC is not subject to RA, although it is moderately sensitive to ClpAP.

Complex restriction enzymes: NTP-driven molecular motors.

Survival is assuredly the prime directive for all living organisms either as individuals or as a species. One of the main challenges encountered by bacterial populations is the danger of bacteriophage attacks, since infection of a single bacterium may rapidly propagate, decimating the entire population. In order to protect themselves against this acute threat, bacteria have developed an array of defence mechanisms, which range from preventing the infection itself via interference with bacteriophage adsorption to the cell surface and prevention of phage DNA injection, to degradation of the injected phage DNA. This last defence mechanism is catalysed by the bacterial restriction-modification (R-M) systems, and in particular, by nucleoside 5'-triphosphate (NTP)-dependent restriction enzymes, e.g. type I and type III R-M systems or the modification-dependent endonucleases. Type I and type III restriction systems have dual properties. They may either act as methylases and protect the host's own DNA against restriction by methylating specific residues, or they catalyse ATP-dependent endonuclease activity so that invading foreign DNA lacking the host-specific methylation is degraded. These defence mechanism systems are further complemented by the presence of methylation-dependent, GTP-dependent endonucleases, that restricts specifically methylated DNA. Although all three types of endonucleases are structurally very different, they share a common functional mechanism. They recognise and bind to specific DNA sequences but do not cleave DNA within those target sites. They belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate DNA so that the subsequent DNA cleavage event occurs at a distance from the endonuclease recognition site. Moreover, DNA cleavage appears to be a random process triggered upon stalling of the DNA translocation process and requiring dimerisation of the bound endonucleases for a concerted break of both DNA strands. In this review, we present a detailed description and analysis of the functional mechanism of the three known NTP-dependent restriction systems: type I and type III restriction-modification enzymes, as well as the methylation-dependent McrBC endonuclease.

New light-sensitive nucleosides for caged DNA strand breaks.

Phototriggered bod cleavage has found wide application in chemistry as well as in biology. Nevertheless, there are only a few methods available for site-specific photochemical induction of DNA strand scission despite numerous potential applications. In this study we report the development of new photocleavable nucleotides based on the photochemistry of o-nitrobenzyl esters. The light-sensitive moieties were generated through introduction of o-nitrophenyl groups at the 5'C position of the nucleoside sugar backbone. The newly synthesized, modified nucleosides were incorporated in oligonucleotides and are able to build stable DNA duplexes. In such a way modified oligonucleotides ca cleaved site-specifically upon irradiation with > 360 nm light with high efficiency. Furthermore, we show that these modifications can be bypassed in DNA synthesis promoted by Thermus aquaticus DNA polymerase.