Single-round deoxyribozyme discovery

Artificial evolution experiments typically use libraries of ∼10¹⁵ sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 10¹⁴ different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs.     

Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn²⁺ as a cofactor, rather than Mg²⁺ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has k_obs on the order of 0.1 min⁻¹, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.     

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn(2+) and Mn(2+) cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4(4)) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N(40) DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.     

Parallel Selections In Vitro Reveal a Preference for 2′–5′ RNA Ligation upon Deoxyribozyme-Mediated Opening of a 2′,3′-Cyclic Phosphate

We previously used in vitro selection to identify Mg²⁺-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5′-hydroxyl group with a 2′,3′-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2′–5′ phosphodiester bonds rather than native 3′–5′ linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2′–5′ linkages. This includes deoxyribozymes with an arrangement that favors 3′–5′ linkages for a different chemical reaction, that of a 2′,3′-diol plus 5′-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2′–5′ RNA linkages upon opening of a 2′,3′-cyclic phosphate with a 5′-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2′,3′-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.[Reviewing Editor: Niles Lehman]     

In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine-pyrimidine junctions

Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited k_obs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ~1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.     

In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg²⁺ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.     

Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection

We previously conducted an in vitro selection experiment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nucleotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short14-15 nt catalytic DNA motif commonly known as 8-17. Herein, we report extensive truncation experiments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8-17 and to determine the effect of excess sequence elements on the activity of this motif and the outcome of selection. Although we observed beneficial, detrimental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2-fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activity, called G15-30, was approximately 2.5-fold larger and experienced a approximately 4-fold greater inhibitory effect from excess sequence elements than the average 8-17 motif. Our results suggest that 8-17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a previously unappreciated selective advantage that may contribute to its widespread recurrence.     

A Comprehensive Strategy to Discover Inhibitors of the Translesion Synthesis DNA Polymerase κ

Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N ²-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N ²-dG DNA–DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.     

Structure-function correlations derived from faster variants of a RNA ligase deoxyribozyme

We previously reported the in vitro selection of several Mg²⁺-dependent deoxyribozymes (DNA enzymes) that synthesize a 2′–5′ RNA linkage from a 2′,3′-cyclic phosphate and a 5′-hydroxyl. Here we subjected the 9A2 deoxyribozyme to re-selection for improved ligation rate. We found two new DNA enzymes (7Z81 and 7Z48) that contain the catalytic core of 7Q10, a previously reported small deoxyribozyme that is unrelated in sequence to 9A2. A third new DNA enzyme (7Z101) is unrelated to either 7Q10 or 9A2. The new 7Z81 and 7Z48 DNA enzymes have ligation rates over an order of magnitude higher than that of 7Q10 itself and they have additional sequence elements that correlate with these faster rates. Our findings provide insight into structure–function relationships of catalytic nucleic acids.     

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K_d) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.     

Multiple occurrences of an efficient self-phosphorylating deoxyribozyme motif

The catalytic and structural characteristics of two new self-phosphorylating deoxyribozymes (referred to as deoxyribozyme kinases), denoted "Dk3" and "Dk4", are compared to those of Dk2, a previously reported deoxyribozyme kinase. All three deoxyribozymes not only utilize GTP as the source of activated phosphate and Mn(II) as the divalent metal cofactor but also share a common secondary structure with significant sequence variations. Multiple Watson-Crick helices are identified within the secondary structure, and these helical interactions confine three extremely conserved sequence elements of 8, 5, and 14 nucleotides in length, presumably for the formation of the catalytic core for GTP binding and the self-phosphorylating reaction. The locations of the conserved regions suggest that these three deoxyribozymes arose independently from in vitro selection. The existence of three sequence variants of the same deoxyribozyme from the same in vitro selection experiment implies that these catalytic DNAs may represent the simplest structural solution for the DNA self-phosphorylation reaction when GTP is used as the substrate.     

Crowding Induces Complex Ergodic Diffusion and Dynamic Elongation of Large DNA Molecules

Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0–40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at ∼20% with an ∼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to ∼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA.     

Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7–11.2-kbp linear duplex DNAs at a rate of ∼250 bp s⁻¹, while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp⁻¹. Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the “leading” AdnB motor propagates a Y-fork by translocation along the 3′ DNA strand, ahead of the “lagging” AdnA motor domain. By tracking the resection of the 5′ and 3′ strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5′ strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3′ strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.     

Aptamer Selection Based on G4-Forming Promoter Region

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as “G4 promoter-derived aptamer selection (G4PAS).” Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K _d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10⁻⁷ M, 6.3 × 10⁻⁹ M, and 4.4 × 10⁻⁷ M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.     

Prospective identification of parasitic sequences in phage display screens

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 10⁹ random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼10⁶ diverse clones prevents the biased selection of parasitic clones.     

Diverse Evolutionary Trajectories Characterize a Community of RNA-Cleaving Deoxyribozymes: A Case Study into the Population Dynamics of In Vitro Selection

Two parallel in vitro selections (denoted Selection A and Selection B) were conducted under different selection-pressure regimes, yielding a diverse community of RNA-cleaving deoxyribozymes. In Selection A, the reaction time was reduced four times (from 5 h to 5 s) over the course of 24 generations, while in Selection B the reaction time was maintained at 5 h for 30 rounds of selective amplification. Sequence alignment was conducted on more than 800 clones assembled from 18 generations that span both selections. Many prominent catalytic sequence classes, including some that extend across both selections, were identified and used to construct fitness landscapes depicting their rise and fall over time. The landscapes from both selections exhibit similar global trends despite differences in population dynamics. Some deoxyribozymes were predominant in the early rounds of selection but gave way to other species that dominated in the middle rounds. Ultimately, these middle classes disappeared from the landscape in favor of new and presumably more fit deoxyribozyme sequence classes. The shape of these landscapes alludes to the presence of many latent deoxyribozymes in the initial library, which can only be accessed by changes in the selection pressure and/or by adaptive mutations. Basic computer simulations provide theoretical corroboration of the experimentally observed pattern of staggered sequence-class transitions across the fitness landscapes. These simulations model the influence of one or more contributing factors, including catalytic rate, folding efficiency, PCR amplification efficiency, and random mutagenesis. This is the first study which thoroughly documents the topography of a deoxyribozyme fitness landscape over many generations of in vitro selection.     

Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection

In vitro selection has been used extensively over the past 10 years to create functionally diverse DNA enzymes. The majority of in vitro selection experiments to date have focused on the outcome rather than the process itself, a process that remains to be fully elucidated. In vitro selection techniques rely on the probability that some DNA molecules in a random-sequence library will fold into an appropriate tertiary structure and catalyze a desired reaction. Thus, sufficient sequence diversity in the DNA pool (and hence more catalytic DNA sequences) is a prerequisite for the successful isolation of efficient deoxyribozymes. The catalytic sequence diversity established by in vitro selection is governed largely by the choice of selection pressures, one of which is the length of the reaction time. The objective of this study was to evaluate the sequence diversity change of a pool of RNA-cleaving deoxyribozymes as a function of the reaction time. Seventeen rounds of in vitro selection were performed, and the reaction time was progressively decreased from 5 h to 5 s. A representative population from each time class was subsequently cloned and sequenced. A decline in sequence diversity was observed with decreasing reaction time, and the relationship appears to be logarithmic. In contrast, a control selection performed with a constant reaction time during each round led to a linear and comparatively very slow decrease in sequence diversity. This study provides the first methodical examination of the change in catalytic sequence diversity that occurs through the course of a deoxyribozyme selection experiment. Moreover, it represents a first step toward fully understanding the intricate pathway that lies between the beginning and end of an in vitro selection experiment.     

Characterization of non-8-17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.     

Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity

Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes—the acceleration of DNA cleavage and the expansion of sequence specificity—are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca²⁺ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme–DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein–DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.     

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo.