RNA aptamer inhibitors of a restriction endonuclease.
ABSTRACT: Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ?12-30 nM) selective competitive inhibitors (IC50 ?20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.
Project description:BACKGROUND: Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of approximately 120-200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. METHODS AND RESULTS: Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but < 400 sites; 69(23.9%) in > 400 but < 700 sites; and the 9(3.1%) enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a nanoparticle for microbicide development and a "recombinant lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII. CONCLUSION: Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.
Project description:The molecular basis of the interaction of KpnI restriction endonuclease (REase) and the corresponding methyltransferase (MTase) at their cognate recognition sequence is investigated using a range of footprinting techniques. DNase I protection analysis with the REase reveals the protection of a 14-18 bp region encompassing the hexanucleotide recognition sequence. The MTase, in contrast, protects a larger region. KpnI REase contacts two adjacent guanine residues and the single adenine residue in both the strands within the recognition sequence 5'-GGTACC-3', inferred by dimethylsulfate (DMS) protection, interference and missing nucleotide interference analysis. In contrast, KpnI MTase does not show elaborate base-specific contacts. Ethylation interference analysis also showed the differential interaction of REase and MTase with phosphate groups of three adjacent bases on both strands within the recognition sequence. The single thymine residue within the sequence is hyper- reactive to the permanganate oxidation, consistent with MTase-induced base flipping. The REase on the other hand does not show any major DNA distortion. The results demonstrate that the differences in the molecular interaction pattern of the two proteins at the same recognition sequence reflect the contrasting chemistry of DNA cleavage and methylation catalyzed by these two dissimilar enzymes, working in combination as constituents of a cellular defense strategy.
Project description:Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5'GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5'-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence-structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence-structure-function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity.
Project description:Most bacterial genomes harbor restriction-modification systems, encoding a REase and its cognate MTase. On attack by a foreign DNA, the REase recognizes it as nonself and subjects it to restriction. Should REases be highly specific for targeting the invading foreign DNA? It is often considered to be the case. However, when bacteria harboring a promiscuous or high-fidelity variant of the REase were challenged with bacteriophages, fitness was maximal under conditions of catalytic promiscuity. We also delineate possible mechanisms by which the REase recognizes the chromosome as self at the noncanonical sites, thereby preventing lethal dsDNA breaks. This study provides a fundamental understanding of how bacteria exploit an existing defense system to gain fitness advantage during a host-parasite coevolutionary "arms race."
Project description:For a very long time, Type II restriction enzymes (REases) have been a paradigm of ORFans: proteins with no detectable similarity to each other and to any other protein in the database, despite common cellular and biochemical function. Crystallographic analyses published until January 2008 provided high-resolution structures for only 28 of 1637 Type II REase sequences available in the Restriction Enzyme database (REBASE). Among these structures, all but two possess catalytic domains with the common PD-(D/E)XK nuclease fold. Two structures are unrelated to the others: R.BfiI exhibits the phospholipase D (PLD) fold, while R.PabI has a new fold termed 'half-pipe'. Thus far, bioinformatic studies supported by site-directed mutagenesis have extended the number of tentatively assigned REase folds to five (now including also GIY-YIG and HNH folds identified earlier in homing endonucleases) and provided structural predictions for dozens of REase sequences without experimentally solved structures. Here, we present a comprehensive study of all Type II REase sequences available in REBASE together with their homologs detectable in the nonredundant and environmental samples databases at the NCBI. We present the summary and critical evaluation of structural assignments and predictions reported earlier, new classification of all REase sequences into families, domain architecture analysis and new predictions of three-dimensional folds. Among 289 experimentally characterized (not putative) Type II REases, whose apparently full-length sequences are available in REBASE, we assign 199 (69%) to contain the PD-(D/E)XK domain. The HNH domain is the second most common, with 24 (8%) members. When putative REases are taken into account, the fraction of PD-(D/E)XK and HNH folds changes to 48% and 30%, respectively. Fifty-six characterized (and 521 predicted) REases remain unassigned to any of the five REase folds identified so far, and may exhibit new architectures. These enzymes are proposed as the most interesting targets for structure determination by high-resolution experimental methods. Our analysis provides the first comprehensive map of sequence-structure relationships among Type II REases and will help to focus the efforts of structural and functional genomics of this large and biotechnologically important class of enzymes.
Project description:Restriction endonucleases (REases) are DNA-cleaving enzymes that have become indispensable tools in molecular biology. Type II REases are highly divergent in sequence despite their common structural core, function and, in some cases, common specificities towards DNA sequences. This makes it difficult to identify and classify them functionally based on sequence, and has hampered the efforts of specificity-engineering. Here, we define novel REase sequence motifs, which extend beyond the PD-(D/E)XK hallmark, and incorporate secondary structure information. The automated search using these motifs is carried out with a newly developed fast regular expression matching algorithm that accommodates long patterns with optional secondary structure constraints. Using this new tool, named Scan2S, motifs derived from REases with specificity towards GATC- and CGGG-containing DNA sequences successfully identify REases of the same specificity. Notably, some of these sequences are not identified by standard sequence detection tools. The new motifs highlight potential specificity-determining positions that do not fully overlap for the GATC- and the CCGG-recognizing REases and are candidates for specificity re-engineering.
Project description:Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.
Project description:RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-kappaB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-kappaB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-kappaB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-kappaB p50(2) and p65(2), respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-kappaB p65(2) in vitro.
Project description:BACKGROUND: Restriction-modification (RM) systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease (REase) is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase (MTase) is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused. RESULTS: The gene for CR.NsoJS138I fusion protein (nsoJS138ICR, from the bacterium Niabella soli) was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene. CONCLUSIONS: Fusions between C proteins and REases have not previously been characterized, though other fusions have (such as between REases and MTases). These results reinforce the evidence for impressive modularity among RM system proteins, and raise important questions about the implications of the C-REase fusions on expression kinetics of these RM systems.
Project description:Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5'-CC/WGG-3' (where W = A/T, and '/' marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5'-CCWGG-3' sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5'-CCWGG-3' sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.