The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

Type I restriction endonucleases are true catalytic enzymes

Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.     

Nicking endonucleases

Nicking endonucleases are a new type of enzymes. Like restriction endonucleases, they recognize short specific DNA sequence and cleave DNA at a fixed position relatively to the recognition sequence. However, unlike restriction endonucleases, nicking endonucleases cleave only one predetermined DNA strand. Until recently, nicking endonucleases were suggested to be naturally mutated restriction endonucleases which had lost their ability to dimerize and as a result the ability to cleave the second strand. We have shown that nicking endonucleases are one of the subunits of heterodimeric restriction endonucleases. Mechanisms used by various restriction endonucleases for double-stranded cleavage, designing of artificial nicking endonucleases on the basis of restriction endonucleases, and application of nicking endonucleases in molecular biology are reviewed.     

Protein footprinting approach to mapping DNA binding sites of two archaeal homing enzymes: evidence for a two-domain protein structure.

The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.     

Type IIE and IIF restriction endonucleases interacting with two recognition sites in DNA

Recently, it was revealed that restriction endonucleases widely used in genetic engineering and molecular biology are diverse not only in DNA sequence specificities but also in mechanisms of their interaction with DNA. In the review type IIE and IIF restriction endonucleases which require the simultaneous interaction with two copies of their recognition sequence for effective hydrolysis of DNA are considered. Crystal structures of these enzymes and their complexes with DNA as well as stepwise interaction with DNA, mechanisms of catalysis and enzyme-mediated DNA looping are discussed. A novel type of DNA-protein recognition was found for type IIE endonucleases when two copies of the same DNA sequence specifically interact with two different amino acid sequences and two structural motifs located in one polypeptide chain.     

Nicking Endonucleases as Unique Tools for Biotechnology and Gene Engineering

Russian Journal of Bioorganic Chemistry - Nicking endonucleases (NE) are a special group of the restriction endonucleases family. These unique enzymes catalyze the hydrolysis of only one DNA strand...     

Cleavage of mispaired heteroduplex DNA substrates by numerous restriction enzymes

The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction enzymes will cleave only perfect cognate recognition sites. In vitro, mispaired heteroduplex DNAs are commonly formed, especially subsequent to polymerase chain reaction amplification. We investigated a panel of restriction endonucleases to determine their ability to cleave mispaired heteroduplex DNA substrates. Two straightforward, non-radioactive assays are used to evaluate mispaired heteroduplex DNA cleavage: a PCR amplification method and an oligonucleotide-based assay. These assays demonstrated that most restriction endonucleases are capable of site-specific double-strand cleavage with heteroduplex mispaired DNA substrates, however, certain mispaired substrates do effectively abrogate cleavage to undetectable levels. These data are consistent with mispaired substrate cleavage previously reported for Eco RI and, importantly, extend our knowledge of mispaired heteroduplex substrate cleavage to 13 additional enzymes.     

Immobilization of restrictases

The search for optimal variants of restriction endonucleases immobilization was begun recently. For some enzymes immobilization was successful due to the presence of covalent bonds on CNBr-sepharose (EcoRI, BamHI, HindIII, TaqI, PaeI, SalI, PvuII). For the enzymes EcoRI, BamHI and HindIII it was due to hydrophobic interaction with triethyl-agarose (triethyl-triphenylmethane). The high yield (up to 80%) of enzymatic activity has been obtained for small number of restriction endonucleases. In the experiments of several amino acid residues modification and immobilization of restriction endonucleases the participation of lysine, arginine, glutamic acid and SH- or S-S-groups in the catalysis and (or) binding of these enzymes with DNA has been shown. The restriction endonucleases immobilization experiments and research of enzymes active centre enrich each other and are very interesting for their use in molecular biology and deepening our knowledge of protein-nucleic interactions.     

A view of an elemental naturalist at the DNA world (Base composition,sequences, methylation)

The pioneering data on base composition and pyrimidine sequences in DNA of pro-and eukaryotes are considered, and their significance for the origin of genosystematics is discussed. The modern views on specificity and functional role of enzymatic DNA methylation in eukaryotes are described. DNA methylation controls all genetic functions and is a mechanism of cellular differentiation and gene silencing. A model of regulation of DNA replication by methylation is suggested. Adenine DNA methylation in higher eukaryotes (higher plants) was first observed, and it was established that one and the same gene can be methylated at both cytosine and adenine moieties. Thus, there are at least two different and seemingly interdependent DNA methylation systems present in eukaryotic cells. The first eukaryotic adenine DNA-methyltransferase is isolated from wheat seedlings and described: the enzyme methylates DNA with formation of N⁶-methyladenine in the sequence TGATCA → TGm⁶ATCA. It is found that higher plants have endonucleases that are dependent on S-adenosyl-L-methionine (SAM) and sensitive to DNA methylation status. Therefore, as in bacteria, plants seem to have a restriction-modification (R-M) system. A system of conjugated up-and down-regulation of SAM-dependent endonucleases by SAM modulations is found in plants. Revelation of an essential role of DNA methylation in regulation of genetic processes is a fundament of materialization of epigenetics and epigenomics. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 12, pp. 1583–1593.     

Diversity of type II restriction endonucleases that require two DNA recognition sites

Orthodox Type IIP restriction endonucleases, which are commonly used in molecular biological work, recognize a single palindromic DNA recognition sequence and cleave within or near this sequence. Several new studies have reported on structural and biochemical peculiarities of restriction endonucleases that differ from the orthodox in that they require two copies of a particular DNA recognition sequence to cleave the DNA. These two sites requiring restriction endonucleases belong to different subtypes of Type II restriction endonucleases, namely Types IIE, IIF and IIS. We compare enzymes of these three types with regard to their DNA recognition and cleavage properties. The simultaneous recognition of two identical DNA sites by these restriction endonucleases ensures that single unmethylated recognition sites do not lead to chromosomal DNA cleavage, and might reflect evolutionary connections to other DNA processing proteins that specifically function with two sites.     

Use of site-specific DNA endonucleases in genome-wide studies of human DNA

During the last decades, site-specific DNA endonucleases have served as a key instrument to study primary structure of DNA and genetic engineering. Here, we describe examples of these enzyme uses in genome-wide analysis of human DNA including restriction endonucleases involvement during sample preparation for sequencing using NGS devices, as well as visualization of cleavage of DNA repeats by endonucleases. The first studies on application of DNA endonucleases in the rapidly developing area of epigenetic analysis of genomes, which is facilitated by the recent discovery of a new class of enzymes, 5-methylcytosinedependent site-specific DNA endonucleases, are of special interest.     

I-BasI and I-HmuI: two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities

I-HmuI and I-BasI are two highly similar nicking DNA endonucleases, which are each encoded by a group I intron inserted into homologous sites within the DNA polymerase genes of Bacillus phages SPO1 and Bastille, respectively. Here, we present a comparison of the DNA specificities and cleavage activities of these enconucleases with homologous target sites. I-BasI has properties that are typical of homing endonucleases, nicking the intron-minus polymerase genes in either host genome, three nucleotides downstream of the intron insertion site. In contrast, I-HmuI nicks both the intron-plus and intron-minus site in its own host genome, but does not act on the target from Bastille phage. Although the enzymes have distinct DNA substrate specificities, both bind to an identical 25bp region of their respective intron-minus DNA polymerase genes surrounding the intron insertion site. The endonucleases appear to interact with the DNA substrates in the downstream exon 2 in a similar manner. However, whereas I-HmuI is known to make its only base-specific contacts within this exon region, structural modeling analyses predict that I-BasI might make specific base contacts both upstream and downstream of the site of intron insertion. The predicted requirement for base-specific contacts in exon 1 for cleavage by I-BasI was confirmed experimentally. This explains the difference in substrate specificities between the two enzymes, including the observation that the former enzyme is relatively insensitive to the presence of an intron upstream of exon 2. These differences are likely a consequence of divergent evolutionary constraints.     

Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity

The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature.     

A highly sensitive selection method for directed evolution of homing endonucleases

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80–100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.     

Restriction map of the temperate phage E105 from the polylysogenic culture of Erwinia carotovora 268

DNA structure of the temperate bacteriophage E105 from polylysogenic culture of Erwinia carotovora 268 has been studied. The viral 29.12-29.17 MD DNA has been shown to be linear and nonpermuted. The complete restriction map of the viral DNA has been constructed for MvaI and HpaI and partial for Eco31 restriction endonucleases based on the pair hydrolysis of the native DNA as well as its fragments. Altogether, 19 sites for restriction endonucleases have been localized on bacteriophage DNA.     

Homing endonuclease structure and function

Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.     

Homing endonuclease I-CreI derivatives with novel DNA target specificities

Homing endonucleases are highly specific enzymes, capable of recognizing and cleaving unique DNA sequences in complex genomes. Since such DNA cleavage events can result in targeted allele-inactivation and/or allele-replacement in vivo, the ability to engineer homing endonucleases matched to specific DNA sequences of interest would enable powerful and precise genome manipulations. We have taken a step-wise genetic approach in analyzing individual homing endonuclease I-CreI protein/DNA contacts, and describe here novel interactions at four distinct target site positions. Crystal structures of two mutant endonucleases reveal the molecular interactions responsible for their altered DNA target specificities. We also combine novel contacts to create an endonuclease with the predicted target specificity. These studies provide important insights into engineering homing endonucleases with novel target specificities, as well as into the evolution of DNA recognition by this fascinating family of proteins.