Characterization of homing endonucleases

Homing endonucleases are a class of site-specific DNA endonucleases encoded by open reading frames within introns and inteins. They initiate the mobility of their host element by recognizing intronless or inteinless alleles of their host gene and making a double-strand break. The homing endonucleases are notable for their long target sites and a tolerance for sequence polymorphisms in their substrates. The methods used to study homing endonucleases are similar to those used to study protein-DNA interactions in general. However, some variations and specialized techniques are useful in characterizing homing endonucleases and these methods are discussed.     

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.     

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.     

Substrate specificity and properties of methyl-directed site-specific DNA endonucleases

Methyl-directed site-specific DNA endonucleases (MD endonucleases) are a small group of enzymes that specifically cleave only methylated DNA. The group includes N6-methyladenine- and 5-methylcytosine-directed enzymes. Although poorly understood, MD endonucleases are of interest for both basic research and application in biotechnology and epigenomics. The review for the first time summarizes the properties of MD endonucleases and considers their role in the bacterial cell and their possible uses in biotechnology and epigenomics.     

Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family

Inteins are genetic elements, inserted in-frame into protein-coding genes, whose products catalyze their removal from the protein precursor via a protein-splicing reaction. Intein domains can be split into two fragments and still ligate their flanks by a trans-protein-splicing reaction. A bioinformatic analysis of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. In each locus, a conserved enzyme coding region is broken in two by a split intein, with a free-standing endonuclease gene inserted in between. Eight types of DNA synthesis and repair enzymes have this ‘fractured’ organization. The new types of naturally split-inteins were analyzed in comparison to known split-inteins. Some loci include apparent gene control elements brought in with the endonuclease gene. A newly predicted homing endonuclease family, related to very-short patch repair (Vsr) endonucleases, was found in half of the loci. These putative homing endonucleases also appear in group-I introns, and as stand-alone inserts in the absence of surrounding intervening sequences. The new fractured genes organization appears to be present mainly in phage, shows how endonucleases can integrate into inteins, and may represent a missing link in the evolution of gene breaking in general, and in the creation of split-inteins in particular.     

SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.     

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.     

真菌线粒体cob中Ⅱ型LAGLIDADG归巢内切酶进化分析

以已公布的114种真菌线粒体基因组数据为依据,对cob内含子及其编码的Ⅱ型LAGLIDADG归巢内切酶进行全面分析,以揭示其进化规律。在cob内含子中共发现27个Ⅱ型LAGLIDADG归巢内切酶基因,其中18个位于S433内含子插入位点,其余9个散布在另外8个插入位点。结合Pfam数据,将Ⅱ型LAGLIDADG归巢内切酶分成10个主要类群,其中4个类群存在不同生物界物种间的水平迁移。S433位点的18个归巢内切酶均属于类群1,它们与宿主内含子可能从共同祖先垂直遗传而来,并在传递过程中伴有水平迁移;其他归巢内切酶及宿主内含子则应是水平迁移的结果。类群1中的归巢内切酶可分为两个亚类,两亚类识别的靶序列存在明显差异;保守模体氨基酸序列分析显示它们大多数具有潜在内切酶活性。全面呈现了真菌线粒体cob内含子及其编码的Ⅱ型LAGLIDADG归巢内切酶的存在状态和进化模式,为归巢内切酶的改造和设计提供了新素材。     

Eight new restriction endonucleases fröm Herpetosiphon giganteus--divergent evolution in a family of enzymes.

Characterization of eight restriction endonucleases isolated from five strains of Herpetosiphon giganteus is described. HgiCI from strain Hpg9 recognizes and cleaves the degenerate sequence: GGPyPuCC, producing 5'-hexanucleotide protruding ends. Endonucleases HgiBI, HgiCII and HgiEI are isoschizomers of AvaII; HgiCIII and HgiDII are isoschizomers of SalI; and HgiDI and HgiGI are isoschizomers of AcyI. Based upon their closely related and in part overlapping recognition specificities a close evolutionary relationship is proposed for all known Hgi restriction endonucleases.     

Analysis of a restriction endonuclease map for a rabbit papillomavirus DNA

A restriction endonuclease map was constructed for rabbit papillomavirus DNA. Analysis of the viral genome by cleavage with specific endonucleases, when compared with bovine papillomavirus types 1 and 2 genome maps, revealed similarities among the three genomes. A number of cleavage sites and their relative order on each genome were found to be conserved.     

Homing endonucleases: keeping the house in order.

Homing endonucleases are rare-cutting enzymes encoded by introns and inteins. They have striking structural and functional properties that distinguish them from restriction enzymes. Nomenclature conventions analogous to those for restriction enzymes have been developed for the homing endonucleases. Recent progress in understanding the structure and function of the four families of homing enzymes is reviewed. Of particular interest are the first reported structures of homing endonucleases of the LAGLIDADG family. The exploitation of the homing enzymes in genome analysis and recombination research is also summarized. Finally, the evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.     

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.     

A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI,TaqII and Tth111II

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5'-ATGAA(N(11/9))-3', was isolated from Thermus sp. DT. TspDTI appears to be a 'twin' of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5'-ACGGA(N(11/9))-3' and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9-100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5-122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对...     

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.     

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.     

Best minimally modified antisense oligonucleotides according to cell nuclease activity.

Minimally modified oligonucleotides belong to the second-generation antisense class. They are phosphodiester oligonucleotides with a minimum of phosphorothioate linkages in order to be protected against serum and cellular exonucleases and endonucleases. They activate RNase H, have weak interactions with proteins, and have thus a better antisense efficiency. Two of them have been designed from an all-phosphorothioate antisense oligonucleotide directed against mdrl-expressing cells. They are protected against serum and cellular enzymatic degradation by the self-forming hairpin d(GCGAAGC) at their 3'-end and by judiciously located phosphorothioate residues, depending on the cellular composition in exonucleases or endonucleases. Besides their already demonstrated ability to cleave pyrimidine sites, endonucleases show some specificity for CpG sites. Their activity is hindered if specific sites are involved in secondary structure as hairpin.