Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions.

The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

Construction of a Z-DNA-Specific Recombinant Nuclease Zαα-FOK for Conformation Studies.

Development of FokI-based engineered nucleases has been a platform technology that enables creation of novel sequence-specific nucleases as well as structure-specific nucleases. Z-DNA-specific nucleases have been constructed by fusing a Z-DNA-binding domain to the nuclease domain of FokI (FN). In particular, Zαα, an engineered Z-DNA-binding domain with a high affinity, is an ideal fusion partner to generate a highly efficient Z-DNA-specific cutter. Here, we describe construction, expression, and purification of Zαα-FOK (Zαα-FN) nuclease in detail. In addition, Z-DNA-specific cleavage is demonstrated by the use of Zαα-FOK.

Axolotl Transgenesis via Injection of I-SceI Meganuclease or Tol2 Transposon System.

The axolotl (Ambystoma mexicanum ) has been widely used as an animal model for studying development and regeneration. In recent decades, the use of genetic engineering to alter gene expression has advanced our knowledge on the fundamental molecular and cellular mechanisms, pointing us to potential therapeutic targets. We present a detailed, step-by-step protocol for axolotl transgenesis using either I-SceI meganuclease or the mini Tol2 transposon system, by injection of purified DNA into one-cell stage eggs. We add useful tips on the site of injection and the viability of the eggs.

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities.

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.

Quantum biology.π-πentanglement signatures in protein-DNA interactions.

The biological functions of DNA are carried out by individual proteins that interact with specific sequences along the DNA in order to prime the molecular processes required by the cellular metabolism. Protein-DNA interactions include DNA replication, gene expression and its regulation, DNA repair, DNA restriction and modification by endonucleases, generally classified as enzymatic functions, or transcription factors functions. To find specific binding target sequences and achieve their aims, in less than one second proteins operate in symbiosis with a crowded cellular environment, identifying extremely small cognate sequences along the DNA chain, which range from 15-20 bps for repressors to 4-6 bps for restriction enzymes. In a previous work, we proposed that the extraordinary ability of proteins to identify consensus sequences on DNA in a short time appears to be dependent on specific quantum signatures such as the entanglement ofπ-πelectrons between DNA nucleotides and protein amino acids, where the couple ofπelectrons function as a radical pair, oneπelectron is located on a specific site of sequence to be identified and the other one performs a quantum walk to identify possible sites of consensus sequence. In this paper, we use the restriction endonucleases enzymes, EcoRV and EcoRI as a case study. These enzymes are able to recognize 3'-GATACT-5' or 3'-GAATCT-5' sequences, respectively. We exploit the analogy of a coin operator with a Bloch sphere to demonstrate that the entanglement betweenπ-πelectrons generated at the contacts on specific GA dimers between proteins and DNA relies on the spin of the electrons that form an initial singlet state. The latter is a maximally entangled state so that the identification of specific nucleotides is associated with the formation of singlet states. On the other hand, during the identification of subsequent GA dimers, the spin-orbit interaction on walkingπelectron induces triplet transitions so that singlet-triplet transitions should manifest an experimentally measurable effect. We propose that the possible experimental evidence of entanglement betweenπ-πelectrons may be due to the phosphorescence signal correspondence to triplet decay processes.

Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains.

Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.

The Influence of 5'R and 5'S cdA and cdG on the Activity of BsmAI and SspI Restriction Enzymes.

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5',8-cyclo-2'-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction-modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme's recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5'S)-5',8-cyclo-2'-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5'R)-5',8-cyclo-2'-deoxyadenosine (RcdA). Interestingly, in the case of 5',8-cyclo-2'-deoxyguanosine (cdG), both 5'S and 5'R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.

Restriction endonuclease T.Smu451I with new cleavage specificity-neoschizomer of T.AsuI.

A specific type II restriction endonuclease T.Smu451I has been purified to electrophoretic homogeneity from the frozen cells of soil bacterium Sphingobacterium multivorum 451 (formerly Flavobacterium multivorum 451), using ultrasonic grinding, nucleic acid removal by streptomycin sulfate, protein precipitation by ammonium sulfate and phosphocellulose P-11, DEAE-Cellulose DE-52, Hepharin-Sepharose CL-6B chromatography, and elucidated several characteristics of T.Smu451I. The molecular weight of the enzyme determined by gel filtration and SDS-polyacrylamide gel electrophoresis was calculated to be 45,000 ± 2000 D (dimer) and 23,000 ± 1000 D (monomer), respectively. The isoelectric point (pI) of T.Smu451I is 5.4. T.Smu451I recognizes pentanucleotide palindromic sequences 5'-GGNC↓C-3' and cleaves between C and C in position shown by arrow to produce 3'-cohesive terminus of trinucleotide. Therefore, T.Smu451I is a neoschizomer of T.AsuI.

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT↓ATC (where ↓ is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Restriction Enzymes.

Restriction enzymes provided the foundation on which molecular cloning was built, and they remain as essential tools in current recombinant DNA technology. The three classes of restriction enzymes and their features are introduced here.

DNA adenine methylase, not the PstI restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli.

We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.

A survey of jaagsiekte sheep retrovirus (JSRV) infection in sheep in the three northeastern provinces of China.

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a "LHMKYXXM" motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the "LHMKYXXM" motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the "LHMKYXXM" motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.

Primordial germ cell expression of SSEA1 and DDX4 (VASA) in female Trichosurus vulpecula (Marsupialia) reveals conserved and unique molecular patterns during marsupial germ cell development.

Development of primordial germ cells (PGCs: precursors to adult gametes) is a key process in vertebrate sexual differentiation. Marsupials are ideal to investigate this phenomenon because much of PGC migration and development unusually occurs postnatally in pouch young. However, investigation of the molecular dynamics underpinning PGC development is restricted to one marsupial model species: the tammar wallaby (Macropus eugenii). Given the reproductive diversity among clades, marsupial PGCs likely exhibit diversity in molecular patterns that could help uncover their developmental dynamics. Here we characterise PGC marker expression (SSEA1 and DDX4) in developing ovaries of the brushtail possum, Trichosurus vulpecula. Female germ cells expressed DDX4 from 6 days postpartum (dpp) and almost all germ cells expressed DDX4 by meiosis (40 dpp), consistent with M. eugenii and eutherian mammals. In contrast, PGCs and oogonia expressed SSEA1 from 12 dpp, throughout proliferation and until entry into meiosis (40-63 dpp). SSEA1 expression was temporally distinct from that of M. eugenii, in which SSEA1 expression persists only until 14 dpp, indicating differential expression between marsupial species at equivalent stages of germ cell development. Hence, the molecular characteristics of M. eugenii germ cells cannot be assumed for all marsupials, as at least one key molecule exhibits species-specific expression.

Protein-DNA target search relies on quantum walk.

Protein-DNA interactions play a fundamental role in all life systems. A critical issue of such interactions is given by the strategy of protein search for specific targets on DNA. The mechanisms by which the protein are able to find relatively small cognate sequences, typically 15-20 base pairs (bps) for repressors, and 4-6 bps for restriction enzymes among the millions of bp of non-specific chromosomal DNA have hardly engaged researchers for decades. Recent experimental studies have generated new insights on the basic processes of protein-DNA interactions evidencing the underlying complex dynamic phenomena involved, which combine three-dimensional and one-dimensional motion along the DNA chain. It has been demonstrated that protein molecules have an extraordinary ability to find the target very quickly on the DNA chain, in some cases, with two orders of magnitude faster than the diffusion limit. This unique property of protein-DNA search mechanism is known as facilitated diffusion. Several theoretical mechanisms have been suggested to describe the origin of facilitated diffusion. However, none of such models currently has the ability to fully describe the protein search strategy. In this paper, we suggest that the ability of proteins to identify consensus sequences on DNA is based on the entanglement of π-π electrons between DNA nucleotides and protein amino acids. The π-π entanglement is based on Quantum Walk (QW), through Coin-position entanglement (CPE). First, the protein identifies a dimer belonging to the consensus sequence, and localize a π on such dimer, hence, the other π electron scans the DNA chain until the sequence is identified. Focusing on the example of recognition of consensus sequences of EcoRV or EcoRI, we will describe the quantum features of QW on protein-DNA complexes during the search strategy, such as walker quadratic spreading on a coherent superposition of different vertices and environment-supported long-time survival probability of the walker. We will employ both discrete- or continuous-time versions of QW. Biased and unbiased classical Random Walk (CRW) have been used for a long time to describe the Protein-DNA search strategy. QW, the quantum version of CRW, has been widely studied for its applications in quantum information applications. In our biological application, the walker (the protein) resides at a vertex in a graph (the DNA structural topology). Differently to CRW, where the walker moves randomly, the quantum walker can hop along the edges in the graph to reach other vertices entering coherently a superposition across different vertices spreading quadratically faster than CRW analogous evidencing the typical speed up features of the QW. When applied to a protein-DNA target search problem, QW gives the possibility to achieve the experimental diffusional motion of proteins over diffusion classical limits experienced along DNA chains exploiting quantum features such as CPE and long-time survival probability supported by the environment. In turn, we come to the conclusion that, under quantum picture, the protein search strategy does not distinguish between one-dimensional (1D) and three-dimensional (3D) cases.

Resection of a DNA Double-Strand Break by Alkaline Gel Electrophoresis and Southern Blotting.

Generation of 3' single-stranded DNA (ssDNA) at the ends of a double-strand break (DSB) is essential to initiate repair by homology-directed mechanisms. Here we describe a Southern blot-based method to visualize the generation of ssDNA at the ends of site-specific DSBs generated in the Saccharomyces cerevisiae genome.

Salt concentration modulates the DNA target search strategy of NdeI.

DNA target search is a key step in cellular transactions that access genomic information. How DNA binding proteins combine 3D diffusion, sliding and hopping into an overall search strategy remains poorly understood. Here we report the use of a single molecule DNA tethering method to characterize the target search kinetics of the type II restriction endonuclease NdeI. The measured search rate depends strongly on DNA length as well as salt concentration. Using roadblocks, we show that there are significant changes in the DNA sliding length over the salt concentrations in our study. To explain our results, we propose a model including cycles of 3D and 1D search in which salt concentration modulates the strategy by varying the length of DNA probed per 1D scan. At low salt NdeI makes a single non-specific encounter with DNA followed by an effective and complete 1D scan. At higher salt, NdeI must execute multiple cycles of target search due to the reduced efficacy of 1D search.

Ash1 and Tup1 dependent repression of the Saccharomyces cerevisiae HO promoter requires activator-dependent nucleosome eviction.

Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.