DNA binding and cleavage by the HNH homing endonuclease I-HmuI

The structure of I-HmuI, which represents the last family of homing endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial colicins, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.     

Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum.

The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene. This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.     

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G₊₄ base pair for the wild-type A:T₊₄ base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T₊₄ were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T₊₄ or the C:G₊₄ base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G₊₄ recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T₊₄ target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G₊₄ target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G₊₄ substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.     

Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.     

I-OmiI and I-OmiII: Two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3′ overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.     

In vivo analysis of the relationships between the splicing and homing activities of a group I intron-encoded I-ScaI/bi2-maturase of Saccharomyces capensis produced in the yeast cytoplasm

The I-ScaI/bi2-maturase of Saccharomyces capensis acts as a specific homing endonuclease promoting intron homing, and as a maturase promoting intron splicing. Using the universal code equivalent of the mitochondrial gene encoding the I-ScaI/bi2-maturase, a number of truncated forms of the synthetic gene were constructed, shortened on either side, as were several mutated alleles of the protein. The shortest translation product that fully retains both activities in vivo corresponds to 228 codons of the C-terminal region of the bi2 intron-encoded protein, whereas proteins resulting from more extensive deletions either at the N-terminus or at the C-terminus (up to 73 and four residues, respectively) were able to complement wholly the lack of endogenous maturase, but all lost the endonuclease activity. Similarly, all introduced mutations completely abolished the I-ScaI activity while some mutant proteins retained substantial splicing function. Immunodetection experiments demonstrated that different cytoplasmically translated forms of the I-ScaI/bi2-maturase protein were imported into mitochondria and correctly processed. They appeared to be tightly associated with mitochondrial membranes. Homology modelling of the I-ScaI/bi2-maturase protein allowed us to relate both enzymatic activities to elements of enzyme structure.     

Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities

Enzymatic activities that cleave Holliday junctions are required for the resolution of recombination intermediates and for the restart of stalled replication forks. Here we show that human cell-free extracts possess two distinct endonucleases that can cleave Holliday junctions. The first cleaves Holliday junctions in a structure- and sequence-specific manner, and associates with an ATP-dependent branch migration activity. Together, these activities promote branch migration/resolution reactions similar to those catalysed by the Escherichia coli RuvABC resolvasome. Like RuvC-mediated resolution, the products can be religated. The second, containing Mus81 protein, cuts Holliday junctions but the products are mostly non-ligatable. Each nuclease has a defined substrate specificity: the branch migration-associated resolvase is highly specific for Holliday junctions, whereas the Mus81-associated endonuclease is one order of magnitude more active upon replication fork and 3'-flap structures. Thus, both nucleases are capable of cutting Holliday junctions formed during recombination or through the regression of stalled replication forks. However, the Mus81-associated endonuclease may play a more direct role in replication fork collapse by catalysing the cleavage of stalled fork structures.     

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.     

Restriction endonucleases HindII and TaqI cleave DNA with mismatched nucleotides within their recognition sequences.

Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double-stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed.     

Restriction endonucleases that bridge and excise two recognition sites from DNA

Most restriction endonucleases bridge two target sites before cleaving DNA: examples include all of the translocating Type I and Type III systems, and many Type II nucleases acting at their sites. A subset of Type II enzymes, the IIB systems, recognise bipartite sequences, like Type I sites, but cut specified phosphodiester bonds near their sites, like Type IIS enzymes. However, they make two double-strand breaks, one either side of the site, to release the recognition sequence on a short DNA fragment; 34 bp long in the case of the archetype, BcgI. It has been suggested that BcgI needs to interact with two recognition sites to cleave DNA but whether this is a general requirement for Type IIB enzymes had yet to be established. Ten Type IIB nucleases were tested against DNA substrates with one or two copies of the requisite sequences. With one exception, they all bridged two sites before cutting the DNA, usually in concerted reactions at both sites. The sites were ideally positioned in cis rather than in trans and were bridged through 3-D space, like Type II enzymes, rather than along the 1-D contour of the DNA, as seen with Type I enzymes. The standard mode of action for the restriction enzymes that excise their recognition sites from DNA thus involves concurrent action at two DNA sites.     

Diatom and chrysophycean cyst profiles in sediment cores from two linked but contrasting Welsh lakes

Llyn Padarn and Llyn Peris are linked by a small river, and although originally a single larger lake, the lakes now support contrasting algal communities. A study of the sequences of diatom remains and chrysophycean cysts present in long sediment cores has been carried out to assess the historical extent of the differences in these algae between the two lakes.

Investigations have included statistical analysis to determine the precision of counting sediment samples. The most efficient sampling and counting procedure, resulting in counts of acceptable precision, involved taking a single sample of sediment from a depth horizon, preparing two replicate slides and counting the entire area of each slide. A comparison of the variation in distribution of these siliceous algae in replicate sediment samples taken from the surface sediment with the variation in distribution down the long sediment core from Llyn Padarn showed that the vertical variation greatly exceeded the horizontal variation. Thus variation in diatom numbers down the sediment core reflects changes in the alga reaching the sediments rather than horizontal patchiness of distribution in the top most layers of sediment.

Studies of the sequences of diatoms and chrysophycean cysts were carried out on a 4·75 m core from Llyn Padarn and 2·1 m core from Llyn Peris representing c. 6000 years and c. 900 years of the two lakes respective history. From c. 6000 until c. 2200 years B.P., Llyn Padarn was an acid oligotrophic lake. It appears that about 2200 years B.P., Llyn Padarn became more enriched, Asterionella formosa was the most numerous diatom preserved in the sediment at this time. Reversion to oligotrophic conditions then occurred c. 2000 years B.P. The oldest material from the Peris sediment core was characterized by centric diatoms similar to those found in Padarn sediment dating from 2000 years B.P. to the surface of the Padarn core. Thus at this time, c. 900 years B.P., the algal communities in the two lakes were similar. Both lakes were oligotrophic. However, by 200 years B.P. the chemistry of the sediment core from Peris showed evidence of extensive copper and slate mining in the environment. At this time the species of siliceous algae preserved in the Peris core changed, and the current differences in algal communities in the two lakes appear to originate from this point in history.     

Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligodeoxynucleotide and enzyme moieties

Class IIS restriction endonucleases cleave double-stranded (ds) DNA at precise distances from their recognition sequences. A method is proposed which utilizes this separation between the recognition site and the cut site to allow a class IIS enzyme, e.g., FokI, to cleave practically any predetermined sequence by combining the enzyme with a properly designed oligodeoxynucleotide adapter. Such an adapter is constructed from the constant recognition site domain (a hairpin containing the ds sequence, e.g., GGATG CCTAC for FokI) and a variable, single-stranded (ss) domain complementary to the ss sequence to be cleaved (at 9 and 13 nucleotides on the paired strands from the recognition sequence in the example of FokI). The ss sequence designated to be cleaved could be provided by ss phage DNA (e.g., M13), gapped ds plasmids, or supercoiled ds plasmids that were alkali denatured and rapidly neutralized. Combination of all three components, namely the class IIS enzyme, the ss DNA target sequence, and the complementing adapter, would result in target DNA cleavage at the specific predetermined site. The target ss DNA could be converted to the precisely cleaved ds DNA by DNA polymerase, utilizing the adapter oligodeoxynucleotide as primer. This novel procedure represents the first example of changing enzyme specificity by synthetic design. A practically unlimited assortment of new restriction specificities could be produced. The method should have many specific and general applications when its numerous ramifications are exploited.     

Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications

Although mobility of the phylogenetically widespread group I introns appears to be mechanistically similar, the phage T4 intron-encoded endonucleases that promote mobility of the td and sunY introns are different from their eukaryotic counterparts. Most notably, they cleave at a distance from the intron insertion sites. The td enzyme was shown to cleave 23-26 nt 5' and the sunY endonuclease 13-15 nt 3' to the intron insertion site to generate 3-nt or 2-nt 3'-OH extensions, respectively. The absolute coconversion of exon markers between the distant cleavage and insertion sites is consistent with the double-strand-break repair model for intron mobility. As a further critical test of the model we have demonstrated that the mobility event is independent of DNA sequences that encode the catalytic intron core structure. Thus, in derivatives in which the lacZ or kanR coding sequences replace the intron, these marker genes are efficiently inserted into intron-minus alleles when the cognate endonuclease is provided in trans. The process is therefore endonuclease-dependent, rather than dependent on the intron per se. These findings, which imply that the endonucleases rather than the introns themselves were the primordial mobile elements, are incorporated into a model for the evolution of mobile introns.     

Crystal structure of BstYI at 1.85A resolution: a thermophilic restriction endonuclease with overlapping specificities to BamHI and BglII

We report here the structure of BstYI, an "intermediate" type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5'-RGATCY-3' (where R=A or G and Y=C or T), cleaving DNA after the 5'-R on each strand to produce four-base (5') staggered ends. The crystal structure of free BstYI was solved at 1.85A resolution by multi-wavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the alpha/beta core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional "arm" substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a "relaxed" DNA specificity.     

Towards a Phylogeny for Coffea (Rubiaceae): identifying well-supported lineages based on nuclear and plastid DNA sequences

BACKGROUND AND AIMS: The phylogenetic relationships between species of Coffea and Psilanthus remain poorly understood, owing to low levels of sequence variation recovered in previous studies, coupled with relatively limited species sampling. In this study, the relationships between Coffea and Psilanthus species are assessed based on substantially increased molecular sequence data and greatly improved species sampling. METHODS: Phylogenetic relationships are assessed using parsimony, with sequence data from four plastid regions [trnL-F intron, trnL-F intergenic spacer (IGS), rpl16 intron and accD-psa1 IGS], and the internal transcribed spacer (ITS) region of nuclear rDNA (ITS 1/5.8S/ITS 2). Supported lineages in Coffea are discussed within the context of geographical correspondence, biogeography, morphology and systematics. KEY RESULTS: Several major lineages with geographical coherence, as identified in previous studies based on smaller data sets, are supported. Other lineages with either geographical or ecological correspondence are recognized for the first time. Coffea subgenus Baracoffea is shown to be monophyletic, but Coffea subgenus Coffea is paraphyletic. Sequence data do not substantiate the monophyly of either Coffea or Psilanthus. Low levels of sequence divergence do not allow detailed resolution of relationships within Coffea, most notably for species of Coffea subgenus Coffea occurring in Madagascar. The origin of C. arabica by recent hybridization between C. canephora and C. eugenioides is supported. Phylogenetic separation resulting from the presence of the Dahomey Gap is inferred based on sequence data from Coffea.     

A model of restriction endonuclease MvaI in complex with DNA: a template for interpretation of experimental data and a guide for specificity engineering

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5'-CCWGG-3' (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5'-CCSGG-3' (S indicates G or C) in the case of R.BcnI, or 5'-CCNGG-3' (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5'-protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence-structure-function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD-(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5'-CCAGG-3' and 5'-CCTGG-3') is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes.     

Expression,purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site

We have overexpressed the type II restriction endonuclease PvuII (R.PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P2₁2₁2₁ with cell constants a = 95.8 Å, b = 86.3 Å, c = 48.5 Å, and diffract X-rays to at least 2.7 Å. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. © 1994 Wiley-Liss, Inc.     

斜纹夜蛾两个天蚕素D基因的克隆及序列分析

天蚕素是昆虫抵御病菌入侵的一类抗菌肽家族。根据斜纹夜蛾Spodoptera litura天蚕素B基因设计特异引物,通过PCR扩增得到2个新的天蚕素基因部分序列,分别命名为cecD1和cecD2（GenBank登录号分别为EF555567和EF555568）。2个基因编码同一个天蚕素D蛋白,该蛋白的成熟肽与天蚕素B存在2个氨基酸残基差异。序列分析发现cecD1和cecD2中分别包含568 bp和377 bp的内含子序列,它们有相同的5′和3′拼接位点,A+T含量分别为59.7%和69.8%,符合大多数真核生物内含子高A+T含量的特征。     

Genetic diversity and population history of two related seabird species based on mitochondrial DNA control region sequences

Geographical variation in two related seabird species, the razorbill (Alca torda) and common guillemot (Uria aalge), was investigated using sequence analysis of mitochondrial DNA (mtDNA) control regions. We determined the nucleotide sequence of the variable 5' segment of the control region in razorbills and common guillemots from breeding colonies across the Atlantic Ocean. The ecology and life history characteristics of razorbill and common guillemot are in many respects similar. They are both considered highly philopatric and have largely overlapping distributions in temperate and subarctic regions of the North Atlantic, yet the species were found to differ widely in the extent and spatial distribution of mtDNA variation. Moreover, the differences in genetic differentiation and diversity were in the opposite direction to that expected from a consideration of traditional classifications and current population sizes. Indices of genetic diversity were highest in razorbill and varied among colonies, as did genotype frequencies, suggestive of restrictions to gene flow. The distribution of genetic variation suggests that razorbills originated from a refugial population in the south-western Atlantic Ocean through sequential founder events and subsequent expansion in the east and north. In common guillemots, genetic diversity was low and there was a lack of geographical structure, consistent with a recent population bottleneck, expansion and gene flow. We suggest that the reduced level of genetic diversity and differentiation in the common guillemot is caused by an inherent propensity for repeated population bottlenecks and concomitantly unstable population structure related to their specialized feeding ecology.