A specificity switch in selected cre recombinase variants is mediated by macromolecular plasticity and water

The basis for the altered DNA specificities of two Cre recombinase variants, obtained by mutation and selection, was revealed by their cocrystal structures. The proteins share similar substitutions but differ in their preferences for the natural LoxP substrate and an engineered substrate that is inactive with wild-type Cre, LoxM7. One variant preferentially recombines LoxM7 and contacts the substituted bases through a hydrated network of novel interlocking protein-DNA contacts. The other variant recognizes both LoxP and LoxM7 utilizing the same DNA backbone contact but different base contacts, facilitated by an unexpected DNA shift. Assisted by water, novel interaction networks can arise from few protein substitutions, suggesting how new DNA binding specificities might evolve. The contributions of macromolecular plasticity and water networks in specific DNA recognition observed here present a challenge for predictive schemes.     

Directed evolution to increase camptothecin sensitivity of human DNA topoisomerase I

BACKGROUND: Human DNA topoisomerase I (top1) relaxes DNA supercoiling during basic nuclear processes. The enzyme is the main target of antitumor agents, such as camptothecins (CPT), that transform top1 into a DNA-damaging agent. RESULTS: By directed evolution of a C-terminal portion, we selected human top1 mutants that were 22-28-fold more CPT-sensitive than wild-type top1 in Saccharomyces cerevisiae cells. The evolved enzymes showed unique mutation patterns and were more processive in plasmid relaxation assays. A top1 mutant had only two amino acid changes in the linker domain, one of which may change a linker/core domain contact surface. The mutant stimulated DNA cleavage to higher levels than the wild-type enzyme and was more sensitive to CPT in a cleavage assay. Moreover, the mutant was more CPT-sensitive than wild-type top1 in a repair-deficient yeast strain. CONCLUSIONS: Mutations in the linker domain can affect DNA binding and CPT sensitivity of human top1. Such drug-hypersensitive topoisomerases may be useful in developing DNA cutters with high cell lethality and in new drug discovery programs.     

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.     

Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme

We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme--an RNA-cleaving catalytic DNA motif previously reported--from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans, and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine tuning when different dinucleotide junctions are given as cleavage sites.     

Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.     

Specificity of the proteasome cleavage to the antigen protein

In the MHC classⅠmolecule binding antigenic peptides processing and presentation pathway,the ubiquitin-proteasome system plays a key role in degrading the protein substrate.For the purpose of studying the specificities of proteasomal cleavage sites,partial least squares method is used to predict the proteasomal cleavage sites,and the predictive accuracy of the model is 82.8%.The specificities of the cleavage sites and the adjacent positions come from the contribution of the amino acids of the samples to the...     

Polymerase evolution: efforts toward expansion of the genetic code

Genetic information is encoded by, but potentially not limited to, a four-letter alphabet. A variety of predominantly hydrophobic nucleobase analogues that form self-pairs in DNA have been examined as third base pair candidates. For example, the PICS self-pair is both stable in duplex DNA and synthesized by some wild-type polymerases with reasonable efficiency. These efforts to expand the genetic code are expected to be facilitated by optimizing both the unnatural nucleobase analogues and the polymerases that replicate them. Here, we report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair. The selection system is based on the co-display on phage of DNA polymerase libraries and a DNA substrate containing the self-pair. Only polymerases that accept the unnatural substrate incorporate a biotin-dUTP to the attached primer and may then be isolated on a streptavidin solid support. A mutant of Sf polymerase, P2, was evolved which both inserts dPICSTP opposite dPICS in the template and extends the unnatural primer terminus by incorporation of the next correct natural dNTP, where the parental enzyme catalyzes neither step at detectable rates. P2 was found to be a triple mutant of Sf, with the mutations F598I, I614F, and Q489H. The evolved properties of P2, as well as the observed mutations, are consistent with an increased affinity for the DNA primer-template containing the self-pair.     

Substrate specificity of the human proteasome

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.     

Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.     

Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site

The bacterial phosphotriesterase has been utilized as a template for the evolution of improved enzymes for the catalytic decomposition of organophosphate nerve agents. A combinatorial library of active site mutants was constructed by randomizing residues His-254, His-257, and Leu-303. The collection of mutant proteins was screened for the ability to hydrolyze a chromogenic analogue of the most toxic stereoisomer of the chemical warfare agent, soman. The mutant H254G/H257W/L303T catalyzed the hydrolysis of the target substrate nearly 3 orders of magnitude faster than the wild-type enzyme. The X-ray crystal structure was solved in the presence and absence of diisopropyl methyl phosphonate. The mutant enzyme was ligated to an additional divalent cation at the active site that was displaced upon the binding of the substrate analogue inhibitor. These studies demonstrate that substantial changes in substrate specificity can be achieved by relatively minor changes to the primary amino acid sequence.     

Alteration of the selectivity of DNA cleavage by a deglycobleomycin analogue containing a trithiazole moiety

The bleomycin (BLM) group of antitumor antibiotics effects DNA cleavage in a sequence-selective manner. Previous studies have indicated that the metal-binding and bithiazole moieties of BLM are both involved in the binding of BLM to DNA. The metal-binding domain is normally the predominant structural element in determining the sequence selectivity of DNA binding, but it has been shown that replacement of the bithiazole moiety with a strong DNA binder can alter the sequence selectivity of DNA binding and cleavage. To further explore the mechanism by which BLM and DNA interact, a trithiazole-containing deglycoBLM analogue was synthesized and tested for its ability to relax supercoiled DNA and cleave linear duplex DNA in a sequence-selective fashion. Also studied was cleavage of a novel RNA substrate. Solid-phase synthesis of the trithiazole deglycoBLM A(5) analogue was achieved using a TentaGel resin containing a Dde linker and elaborated from five key intermediates. The ability of the resulting BLM analogue to relax supercoiled DNA was largely unaffected by introduction of the additional thiazole moiety. Remarkably, while no new sites of DNA cleavage were observed for this analogue, there was a strong preference for cleavage at two 5'-GT-3' sites when a 5'-(32)P end-labeled DNA duplex was used as a substrate. The alteration of sequence selectivity of cleavage was accompanied by some decrease in the potency of DNA cleavage, albeit without a dramatic diminution. In common with BLM, the trithiazole analogue of deglycoBLM A(5) effected both hydrolytic cleavage of RNA in the absence of added metal ion and oxidative cleavage in the presence of Fe(2+) and O(2). In comparison with BLM A(5), the relative efficiencies of hydrolytic cleavage at individual sites were altered.     

Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites

An approach for multiparallel target identification and relative quantification of in vitro kinase activities in two different biological samples, using liquid chromatography/mass spectrometry (LC/MS), is described. Synthetic target peptides, containing the putative regulatory phosphorylation sites of sucrose-phosphate synthase (SPS) isoenzymes from Arabidopsis thaliana, were simultaneously in vitro phosphorylated and their phosphorylation states determined. Quantification was achieved by stable isotope labeling of the phosphoserine moiety with ethanethiol and [(2)D(5)]-ethanethiol. This revealed different kinase activities in extracts of wild-type (WT) plants and mutant plants lacking plastidic phosphoglucomutase (PGM). The multiparallel assay allowed the determination of favored substrate specificities among the putative phosphorylation sites in SPS. Additionally, we extended the method to unambiguously identify phosphorylation sites in peptides via differential labeling.     

Novel strategy for the design of a new zinc finger: creation of a zinc finger for the AT-rich sequence by alpha-helix substitution

In this communication, a novel strategy for the design of a zinc finger peptide on the basis of alpha-helix substitution has been demonstrated. Sp1HM is a helix-substituted mutant for the wild-type Sp1(zf123) and its alpha-helix of each finger is replaced by that of fingers 4-6 of CF2-II. The circular dichroism spectrum of Sp1HM suggests that Sp1HM has an ordered secondary structure similar to that of Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that Sp1HM specifically binds to the AT-rich sequence (5'-GTA TAT ATA-3') with 3.2 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the alpha-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.     

An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.     

Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate,the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted altematively regularly and the plasmid of pGEM-MDRI/MRPI used to transcribe the M DRI/MRPI(196/210) substrate containing double target sites was also constructed by DNA recombination.Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct.The cleavage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system.The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively,and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively.The multiribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]5 is more significantly superior to that of the [Coat'A 196Rz/Coat'B210Rz]1 in cleavage of RNA substrate.The fractions cleaved by [Coat'A196Rz/Coat'B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference.The studies of Mg2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg2+ ions concentration.The plot of Ig(kobs) vs.Igc(Mg2+) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg2..It suggests that Mg2+ ions play a crucial role in multi-ribozyme cleavage on the substrate.     

Intracellular Detection and Evolution of Site-Specific Proteases Using a Genetic Selection System

Development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. Difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as therapeutics. Here, we disclose a genetic system, designed to report on site-specific proteolysis through the survival of a bacterial host, and the implementation of this method in the directed evolution of proteases with a non-native substrate preference. The high sensitivity potential of this system was established by monitoring the activity of the Tobacco Etch Virus protease (TEV-Pr) against co-expressed substrates of various recognition level and corroborated by both intracellular and cell-free assays. The genetic selection system was then used in an iterative mode with a library of TEV-Pr mutants to direct the emergence of proteases favoring a nominally poor substrate of the stringently selective protease. The retrieval of mutant enzymes displaying enhanced proteolytic properties against the non-native sequence combined with reduced recognition of the cognate hexapeptide substrate demonstrates the potential of this system for evolving proteases with improved or completely unprecedented properties.     

Amplified nucleic acid sensing using programmed self-cleaving DNAzyme

A newly designed target-assisted self-cleavage (TASC) probe composed of a target-binding site and a DNAzyme domain undergoes TASC when activated via hybridization with a target DNA/RNA. This self-splicing or self-dissociation reaction occurs in a catalytic manner with the probe as a substrate and the target as a catalyst, since the fragmented products are automatically released from the target, thus amplifying the sequence information of the latter under non-PCR, i.e., isothermal and enzyme/reagent-free, conditions. A fluorescence-reporting TASC probe having a fluorescein/dabsyl FRET pair across the cleavage site allows a mix-and-read discrimination of single-nucleotide differences in the target.     

Effect of polymethylene and phenylene linking groups on the DNA cleavage specificity of distamycin-linked hydroxamic acid-vanadyl complexes

Two types of distamycin-linked hydroxamic acids (DHA), which contain various lengths of polymethylene chains (PM-DHA) and relatively rigid phenylene ones (Ph-DHA), have been synthesized for the first time. Their DNA cleavage specificities were investigated by an end-labeled fragment cleavage experiment in the presence of vanadyl ion and hydrogen peroxide. The DNA cleavage by the PM-DHA x VO(II) complexes was shown to be very dependent on the length of the chain and the AT sequences. The tetramethylene DHA (1b) complex exhibited highly specific cleavage patterns flanking the 8 and 10 AT sites. Interestingly, the Ph-DHA complexes selectively cleaved the 5' end-labeled strand at the AT sites, but did not cleave the 3' end-labeled strand. The vanadyl complexing moieties and the local sequence conformation of the AT tract are suggested to contribute significantly to the DNA recognition of the PM-DHA x VO(II) complexes.