Academic literature on the topic 'DNA-directed DNA polymerase'

ABSTRACT DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol μ), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol μ has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol β, although pol μ's substrate specificity is similar to that of pol β in most other respects. Moreover, pol μ more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol μ frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.

The modified nucleotides, N2-(p-n-butylphenyl)dGTP and 2-(p-n-butylanilino) dATP and related compounds have been developed as inhibitor-probes of B family DNA polymerases. Synthetic approaches to these compounds are summarized. The nucleotides are potent, non-substrate inhibitors of DNA polymerase a. In contrast, they inhibit other members of the family with less potency but act as substrates for these enzymes. Modelling of the inhibitor: enzyme binding mechanism has been done based on the known structure of E. coli DNA polymerase I, and site-directed mutagenesis experiments to evaluate this mechanism are proposed.

ABSTRACT DNA synthesis and DNA polymerase-α, -β and -γ activities in the rabbit mammary gland were studied during hormone-directed cellular growth. It was found that during pregnancy, early lactation and after injection of prolactin, changes in the activity of DNA polymerase-α paralleled the rate of mammary gland DNA synthesis. It was also found that the amount of polymerase-α activity bound to isolated chromatin depended on the physiological state of the animal. During pregnancy and early lactation changes in the activity of chromatin-bound enzyme correlated directly with the rate of DNA synthesis (r = 0·83). Moreover, in virgin rabbits treated with prolactin the activity of chromatin-bound DNA polymerase-α increased markedly at the same time as the DNA-synthetic rate increased. No correlation of the DNA-synthetic rate was found with the activity of soluble (cytosolic) DNA polymerase-α or with the activity of soluble or chromatin-bound DNA polymerases-β and -γ. On the basis of these results it is suggested that in the developing mammary gland both the activity and cellular distribution of DNA polymerase-α might be subject to hormonal regulation. J. Endocr. (1987) 114, 139–145

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3′-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3′-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3′→P5′ phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3′-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

Abstract DNA replication is a process in which a cell duplicates its genome before cell division, and must proceed accurately and in organized manner to guarantee maintenance of the integrity of the genetic information. DNA polymerases are enzymes that catalyse the synthesis of the new DNA strand by utilizing the parental strand as a template. In addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also needed in other processes like DNA repair and DNA recombination. The DNA polymerase is an essential DNA polymerase in eukaryotes and is required for chromosomal DNA replication. It has also been implicated in DNA repair, recombination, and in transcriptional and cell cycle control. The regulation of the human enzyme was explored by analysing its expression, phosphorylation and protein-protein interactions. Expression of both the A and B subunits of the human DNA polymerase ε was strongly growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA levels were up-regulated at least 5-fold. In actively cycling cells, however, the steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in G1/S phase. The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107 was demonstrated in vitro. A down-regulation element, located immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription factors. A model of the promoter function is presented. Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to Saccharomyces cerevisiae Dpb11, Schizosaccharomyces pombe Cut5, Drosophila melanogaster Mus101 and the human Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose expression is induced at the G1/S border and is required for chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci, which do not represent sites of ongoing DNA replication. However, if DNA is damaged or replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating cell nuclear antigen (PCNA) containing foci that represent stalled replication forks. Finally, phosphorylation of DNA polymerase ε was described and at least three immunologically distinct and differentially phosphorylated forms were shown to exist. Phosphorylation is on serine and threonine residues and shows a cell cycle dependent fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1 co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In contrast, TopBP1 was shown to be associated with a hyperphosphorylated form.

This thesis is divided into three parts, united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol δ). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs, selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol α) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol δ, and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol δ inhibitor on cellular DNA synthesis. Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA), and related compounds, of Chinese hamster ovary (CHO) cell ([3H]thymidine [3H]TdR) incorporation, was investigated. The potency of the compound could largely be explained by its potency (IC50 = 23 μM) as an inhibitor of CHO cell [3H]TdR uptake. BuAdA inhibited incorporation by CHO cells of [32p]phosphate into DNA relatively weakly, displaying an IC50value of 80 μM. Differential inhibition of DNA polymerases alpha and delta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol δ derived from calf thymus selectively with respect to pol α derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol δ weakly, but with greater potency than pol α. Based on this lead, an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol δ with a potency (Ki = 1.8 μM) twenty-two times greater than that displayed for pol α. Kinetic studies indicated that COMDP inhibited pol δ competitively with the dNTP specified by the template, but not competitively with the template:primer. Analogous experiments with pol α showed that the compound inhibited that enzyme uncompetitively with the dNTP, and not competitively with the template:primer. COMDP was a weak inhibitor of the 3' → 5' exonuclease activity of pol δ, displaying an IC50value greater than 1 mM. Inhibition of permeabilized cell DNA synthesis bv a selective pol δ inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (IC50= 200 μM) did not clearly indicate the participation of pol δ in cellular DNA replication. Prospectus. The thesis concludes with a prospectus for the development of pol δ inhibitors with improved properties compared to COMDP.

The thermophilic enzyme, Thermus aquaticus (Taq) DNA polymerase, is an essential tool in molecular biology because of its ability to synthesis DNA in vitro and its inherent thermal stability. Taq DNA polymerase is widely used in the polymerase chain reaction (PCR), an essential technique in a broad range of different fields from academic research to clinical diagnostics. The use of PCR-based tests in diagnostic testing is ever increasing; however, many of the samples being tested contain substances that inhibit PCR and prevent target amplification. Many attempts have been made to engineer polymerases not only to increase resistance to overcome the problem of inhibition, but also to enhance other characteristics such as fidelity, processivity and thermostability. Heparin, found in blood samples, and phytate, found in faecal samples, are two examples from a number of known PCR inhibitors. The mode of action of most PCR inhibitors is not well understood, but inhibition is thought to occur by enzyme binding or through the chelation of Mg2+ ions essential for PCR. In this project, a system of directed evolution by compartmentalised self-replication (CSR) was established and successfully employed to screen a mutant library for Taq DNA polymerase variants with enhanced resistance to the inhibitors heparin and phytate. CSR is a recently-established high-throughput method for the creation of novel polymerases, based on a feedback loop whereby polymerase variants replicate their own encoding gene. A mutant library of 106 variants was produced by random mutagenesis error-prone PCR, in which only the polymerase domain of Taq was mutagenised. Firstly, the CSR system was established and tested by performing a screen in the presence of heparin to select for heparin-resistant variants. Characterisation of selected variants revealed that a single round of CSR had produced a Taq variant (P550S, T588S) with a 4-fold increase in heparin resistance. The IC50 was increased from 0.012U/ml heparin to 0.050U/ml heparin. The study with heparin was followed by a phytate screen, in which two rounds of CSR were performed with an initial round of error-prone PCR followed by re-diversification (recombination) of the mutant library using the staggered extension process (StEP). The two rounds of CSR yielded a Taq variant with a 2-fold increase in phytate-resistance compared to the wild-type, with IC50 increased from 360μM phytate to 700μM phytate. The best phytate mutant (P685S, M761V, A814T) was further characterised and it was found that the catalytic activity, thermostability and fidelity of the mutant were comparable to the wildtype enzyme. The position of resistance-conferring mutations of the novel Taq variants evolved in this study provided some evidence for the inhibitors’ predicted modes of action in the case 2 of both phytate and heparin. As phytate’s mode of action is poorly understood, further investigations were performed to elucidate its role in PCR inhibition. A thorough investigation into the importance of relative phytate and Mg2+ levels on PCR was conducted and revealed for the first time convincing evidence that the primary mode of phytatemediated PCR inhibition is by chelation. Further work led to the successful crystallisation of Taq in the presence of phytate, although subsequent X-ray diffraction data to 2.5Å did not reveal phytate bound within the enzyme structure. Site-directed mutagenesis studies were used to probe cross-over between heparin and phytate-conferring mutations. Thus, in addition to providing valuable information for novel Taq variants with a potential application in fecal-based PCR diagnostic tests, this project has begun to provide insight into the fundamental aspects of the mode of action of phytate as a polymerase and PCR inhibitor.

Les inhibiteurs nucléosidiques de la transcriptase inverse (INTIs) sont des médicaments anti-VIH pouvant induire des lipodystrophies. Le but de cette thèse a été d'évaluer les effets mitochondriaux et métaboliques des INTIs (AZT, d4T, ddC,ddI, 3TC chez la souris. Après deux semaines de traitement, seuls les analogues de la thymide (AZT et d4T) augmentaient l'oxydation hépatique des acides gras et les corps cétoniques plasmatiques, sans induire un dysfonctionnement mitochondrial profond. Ces effets étaient reproduits par l'acide β-aminoisobutyrique (BAIBA), un catabolite de la thymine. Après six semaines, seuls le d4T, l'AZT et le BAIBA augmentaient les corps cétoniques et diminuaient la masse grasse corporelle des souris Swiss,sans dépléter l' ADNmt dans le tissu.
Nucleoside reverse-transcriptase inhibitors (NRTIs), used in the treatment of HIV infection can induce lipodystrophy. The aim of this work was to better characterize the mitochondrial and metabolic effects induced by NRTIs (AZT, d4T, ddC, ddI, 3TC) in mice. The thymidine analogs AZT and d4T increased fatty acids oxidation in liver and plasma ketone bodies after 2 weeks of treatment, without a profund mitochondrial dysfunction. These effects were reproduced by β-aminoisobutyric acid (BAIBA), a thymine catabolite. The derivatives d4T, AZT and BAIBA still increased ketone bodies and decreased body fat mass after 6 weeks of treatment in Swiss mice. However, mitochondrial DNA was not decreased in epididymal white adipose tissue and glucido-lipidic metabolism was unchanged

Les adénovirus humains (HAdV) sont responsables d’une importante morbidité et mortalité chez les patients receveurs de cellules souches hématopoïétiques (CSH), en particulier chez les enfants. Le brincidofovir (BCV) prodrogue du cidofovir est un analogue de la cytosine. Il inhibe l’activité de l’ADN polymérase (ADN pol) des HAdV par arrêt de l’élongation et compétition avec les nucléotides naturels. Les objectifs de ce travail sont d’étudier la diversité génétique de l’ADN pol des HAdV et de mettre en place des outils pour l’analyse de la sensibilité des HAdV C et de résistance aux BCV. Dans une première partie, nous avons réalisé le séquençage complet du gène ADN pol de 60 souches virales cliniques. Les résultats montrent une variabilité nucléotidique de 9,9% et peptidique de 8,7%, variable en fonction du type. La région la plus variable est la région NH2 terminale avec 44,2% des mutations d’acides aminés. Aucune des mutations décrites comme associées à la résistance au CDV ou au BCV n’a été détectée. L’analyse du polymorphisme a aussi montré que les séquences ADN pol permettent d’identifier les sources de transmission au cours d’une épidémie. Enfin nos observations suggèrent la faible probabilité d’évènements de recombinaison comme facteur d’évolution du gène codant pour l’ADN pol. Dans une seconde partie, nous avons développé une stratégie de génétique inverse à partir de deux plasmides, un codant pour le génome entier délété de l’ADN pol (Adv-deltaPol) et un codant uniquement pour l’ADN pol (Pol) (3 kilobase) dans lequel les mutations sont introduites par mutagénèse dirigée (Pol-muté). Après une étape de recombinaison bactérienne en un seul plasmide (Adv-deltaPol-Pol-muté), des cellules 293T sont transfectées afin de produire les virus recombinants. La susceptibilité au BCV des virus mutés sera analysée sur des cellules A549. Au total de 12 plasmides Pol-muté ont été produits, le virus recombinant L677F a été obtenu. Les productions des autres virus recombinants sont en cours
Human adenoviruses (HAdV) are responsible for significant morbidity and mortality in hematopoietic stem cell (HSC) recipients, particularly in children.Brincidofovir (BCV) prodrug of cidofovir is an analogue of cytosine. It inhibits the DNA polymerase (DNA pol) activity of HAdV by stopping elongation and competition with natural nucleotides.The objectives of this work are to study the genetic diversity of the pol DNA of HAdV and to set up tools for the analysis of HAdV C sensitivity and resistance to BCV.In a first part, we carried out the complete sequencing of the DNA pol gene of 60 clinical viral strains. The results show a nucleotide variability of 9.9% and peptide variability of 8.7%, variable according to the type. The most variable region is the NH2 terminal region with 44.2% of the amino acid mutations. None of the mutations described as associated with CDV or BCV resistance were detected. Polymorphism analysis has also shown that DNA pol sequences can identify sources of transmission during an outbreak. Finally, our observations suggest the low probability of recombination events as a factor of evolution of the gene coding for the DNA pol. In a second part, we developed a reverse genetic strategy from two plasmids, one coding for the whole genome deleted from the DNA pol (Adv-deltaPol) and one coding only for the viral DNA pol (Pol). 3 kilobase) in which the mutations are introduced by site-directed mutagenesis (Pol-mutated). After a bacterial recombination step into a single plasmid (Adv-deltaPol-Pol-mutated), 293T cells are transfected to produce the recombinant viruses. BCV susceptibility of mutated viruses will be analyzed on A549 cells. A total of 12 Pol-mutated plasmids were produced, recombinant virus L677F was obtained. Production of other recombinant viruses is ongoing

Oligonucleotides enable many biotechnological applications; however they are easily degraded by nucleases. Many nucleotides modified at the 2’ position are degraded at decreased rates which improves oligonucleotide utility. Most applications of oligonucleotides rely on enzymatic synthesis. Unfortunately, native DNA polymerases do not recognize most useful modified nucleotide substrates. Directed evolution has been used to identify mutants of Taq DNA polymerase I (Taq) that recognize substrates with 2’ modifications. While mutant enzymes capable of modified nucleotide addition have been identified, to date, all of these enzymes are limited by their inability to synthesize full length modified DNA. Despite considerable efforts to evolve new activity there has been little work done to quantitatively characterize these evolved enzymes. This thesis work presents efforts to synthesize modified primers that will help comparatively and quantitatively characterize three enzymes previously evolved to recognize 2’ modified substrates. Using the methods developed in this thesis project, our lab will be able to characterize the relationship between the number of modified nucleotides in the primer terminus and the rate of modified and unmodified nucleotide addition. Future work will identify key enzymatic steps that limit extension in these enzymes with implications for the future design of Taq mutants capable of synthesizing long 2’ modified oligonucleotides.

AbstractThe unique specificity of DNA interactions and our ability to synthesize artificial functionalized DNA sequences makes it the ideal material for controlling self-assembly and chemical reactions of components attached to DNA sequences. Inspired by the field of molecular electronics and the lack of methods to assemble molecular components, we have explored the organization of conjugated molecular components using DNA-based self-assembly. In this chapter, we provide an overview of our efforts first to assemble and chemically couple conjugated molecules directed by DNA, and more recently to assemble conjugated polymers in DNA nanostructures. At the end of the chapter, we provide a short overview of work by other groups in the field.

Abstract Oligonucleotide directed in vitro mutagenesis is a widely used procedure for the study of the structure and function of DNA and the protein for which it codes, for studies on regulation of gene expression and DNA replication, and for modification of food and therapeutic proteins. A wide variety of techniques is available for performing in vitro mutagenesis (reviewed in ref. 1 and this book). One strategy is to clone the segment of DNA to be mutated into a vector whose DNA exists in both single and double-stranded forms. An oligonucleotide complementary to the region to be altered, except for a limited internal mismatch, is hybridized to a single-strand copy of the DNA. A complementary strand is then synthesized by DNA polymerase using the oligonucleotide as primer. Ligase is used to seal the new strand to the 5’-end of the oligonucleotide. The double-stranded DNA, homologous except for the intended mutation, is then transformed into E. coli, resulting in two classes of progeny; the parental and those carrying the oligonucleotide directed mutation. Since there are both parental and mutant progeny, no more than half of the progeny will be mutant. In practice, a much lower fraction is usually obtained. Recently, a number of methods have been developed which are more efficient. These methods create an asymmetry between the two strands of the heteroduplex and permit selection against the wild-type strand (see also Chapters 1 and 3).

Abstract Over the last decade, a variety of efficient and reliable methods have been developed for the construction of site-directed mutations in DNA using synthetic oligonucleotides (reviewed in refs 1-3). This has revolutionized the study of gene regulation and protein -structure and function. All of these mutagenesis methods fall into one of three broad strategies. One approach uses an oligonucleotide complimentary to part of a single-stranded (ss) DNA template but containing an internal mismatch(es) to direct the desired mutation (see Section 1.1, and Chapters 2 to 5 and 9). A second strategy is to replace the region of interest with a synthetic mutant fragment generated by annealing complimentary oligonudeotides (‘cassette mutagenesis’ (ref. 4 and Chapter 10)), or by hybridization and ligation of a number of oligonucleotides (reviewed in ref. 5). The third and most recent strategy, has been to harness the power of the polymerase chain reaction (PCR) (6) to generate a mutant fragment starting from a double-stranded (ds) DNA template using mismatched oligonucleotides (refs 7 and 8, and Chapter 11). In this chapter an efficient method of site-directed mutagenesis using mismatched oligonucleotides (first strategy above) is discussed.

DNA assembly has always been a vital foundational technology for synthetic biology and other bioengineering processes. The development of restriction digestion and ligation methods has played a significant role in developing better DNA assembly methods with higher fidelity, efficiency, and modularity. Daniel Gibson discovered an exonuclease-based robust cloning method to assemble the DNA in the correct sequence known as the Gibson assembly. This method has been widely adopted as a major workhouse for synthetic biology experiments that have provided a new platform for the rapid cloning of multiple DNA fragments into the vector with a minimal time frame under isothermal conditions utilizing an overhang generated by 5' exonuclease, polymerase, and a DNA ligase. The Gibson assembly technique is widely applied in assembling single fragments, multiple fragments, site-directed mutagenesis, shotgun cloning, and creating library construct. Several advancements were observed in terms of developing minimal cells and synthesizing the cells and combining them with CRISPR technology.

Abstract Phage display is a process by which a peptide or a protein is expressed as an exterior fusion to a surface protein of a phage particle. The peptide or protein sequence can be deduced from its encoding DNA sequence that resides in the phage particle or in a transductant. Amplification of the DNA of interest can take place by phage/transductant propagation or by polymerase chain reaction (PCR). By producing large populations of phage particles, each expressing a unique peptide or protein, peptide/protein libraries can be obtained. Peptides or proteins, interacting with defined molecular targets most often proteins can be isolated from such libraries by enrichment through repeated cycles of panning. Hence, phage display can be thought of as a ‘‘search engine’’ of protein^target interactions. The pioneering work of Smith (113) first demonstrated surface display of peptides in filamentous phage fd. This innovation was extended to peptide libraries of fd and M13 (19, 22, 108) and phagemid display was introduced (6). The display of proteins such as antibody domains and combinatorial antibody libraries soon followed (5, 59, 75). From the beginning of the 1990s phage-display-related publications have grown exponentially (128). Several reviews (some general, e.g., 58, 125; and many specialized) are available. There are numerous reports and several laboratory manuals (4) describing development and use of filamentous phage display in identification of peptide or protein interactions with simple organic compounds, antibodies, receptors, etc. Phage display is also a useful tool in protein engineering and directed evolution (44). Then there is the large sector of phage antibody display (64) and the more recent field of immune profiling and its implication for vaccine development (15, 112). Furthermore, complex targets such as cells (92) and whole tissues/organs (91) have been subjected to phage display analysis. These studies explore novel approaches for in vivo homing in gene/drug delivery (78), cancer surveillance/treatment (2, 86, 103), and imaging (127).

Abstract The polymerase chain reaction makes possible a new method for engineering genes and is useful for both mutagenesis and recombination of sequences. This approach has significant advantages over current techniques because it is fast, straightforward, and not limited by the need for restriction sites. The development of recombinant DNA technology can be divided into three generations. The first generation approaches take advantage of natural recombination mechanisms in vivo to map and study genes. Into this category would fall classical genetic linkage analysis, and the use of mechanisms for homologous recombination between phages, for example, to generate sets of random recombinants between related genes. The second generation began with the use of restriction endonucleases and DNA ligase to specifically take genes apart, and put them back together in new combinations in vitro. It was the advent of this technology that made the field of molecular genetics what it is today.

Objectives: Our BARD proposal on the impact of RNA-dependent RNA polymerase 1 (RDR1) in plant defense against viruses was divided into four original objectives. 1. To examine whether a high level of dsRNA expression can stimulate RDR1 transcription independent of salicylic acid (SA) concentration. 2. To determine whether the high or low level of RDR1 transcript accumulation observed in virus resistant and susceptible cultivars is associated with viral resistance and susceptibility. 3. To define the biogenesis and function of RDR1-dependent endogenous siRNAs. 4. To understand why Cucumber mosaic virus (CMV) can overcome RDR1-dependent resistance. The objectives were slightly changed due to the unique finding that cucumber has four different RDR1 genes. Background to the topic: RDR1 is a key plant defense against viruses. RDR1 is induced by virus infection and produces viral and plant dsRNAs which are processed by DICERs to siRNAs. siRNAs guide specific viral and plant RNA cleavage or serve as primers for secondary amplification of viral-dsRNA by RDR. The proposal is based on our preliminary results that a. the association of siRNA and RDR1 accumulation with multiple virus resistance, and b. that virus infection induced the RDR1-dependent production of a new class of endogenous siRNAs. However, the precise mechanisms underlying RDR1 induction and siRNA biogenesis due to virus infection remain to be discovered in plants. Major conclusions, solutions and achievements: We found that in the cucurbit family (cucumber, melon, squash, watermelon) there are 3-4 RDR1 genes not documented in other plant families. This important finding required a change in the emphasis of our objectives. We characterized 4 RDR1s in cucumber and 3 in melon. We demonstrated that in cucumber RDR1b is apparently a new broad spectrum virus resistance gene, independent of SA. In melon RDR1b is truncated, and therefore is assumed to be the reason that melon is highly susceptible to many viruses. RDR1c is dramatically induced due to DNA and RNA virus infection, and inhibition of RDR1c expression led to increased virus accumulation which suggested its important on gene silencing/defense mechanism. We show that induction of antiviral RNAi in Arabidopsis is associated with production of a genetically distinct class of virus-activated siRNAs (vasiRNAs) by RNA dependent RNA polymerase-1 targeting hundreds of host genes for RNA silencing by Argonaute-2. Production of vasiRNAs is induced by viruses from two different super groups of RNA virus families, targeted for inhibition by CMV, and correlated with virus resistance independently of viral siRNAs. We propose that antiviral RNAi activate broad-spectrum antiviral activity via widespread silencing of host genes directed by vasiRNAs, in addition to specific antiviral defense Implications both scientific and agricultural: The RDR1b (resistance) gene can now be used as a transcription marker for broad virus resistance. The discovery of vasiRNAs expands the repertoire of siRNAs and suggests that the siRNA-processing activity of Dicer proteins may play a more important role in the regulation of plant and animal gene expression than is currently known. We assume that precise screening of the vasiRNA host targets will lead in the near future for identification of plant genes associate with virus diseases and perhaps other pathogens.

The Genetic Technology (Precision Breeding) Act (England) aims to develop a science-based process for the regulation and authorisation of precision bred organisms (PBOs). PBOs are created by genetic technologies but exhibit changes which could have occurred through traditional processes. This current review, commissioned by the Food Standards Agency (FSA), aims to clarify existing terminologies, explore viable methods for the detection, identification, and quantification of products of precision breeding techniques, address and identify potential solutions to the analytical challenges presented, and provide recommendations for working towards an infrastructure to support detection of precision bred products in the future. The review includes a summary of the terminology in relation to analytical approaches for detection of precision bred products. A harmonised set of terminology contributes towards promoting further understanding of the common terms used in genome editing. A review of the current state of the art of potential methods for the detection, identification and quantification of precision bred products in the UK, has been provided. Parallels are drawn with the evolution of synergistic analytical approaches for the detection of Genetically Modified Organisms (GMOs), where molecular biology techniques are used to detect DNA sequence changes in an organism’s genome. The scope and limitations of targeted and untargeted methods are summarised. Current scientific opinion supports that modern molecular biology techniques (i.e., quantitative real-time Polymerase Chain Reaction (qPCR), digital PCR (dPCR) and Next Generation Sequencing (NGS)) have the technical capability to detect small alterations in an organism’s genome, given specific prerequisites of a priori information on the DNA sequence of interest and of the associated flanking regions. These techniques also provide the best infra-structure for developing potential approaches for detection of PBOs. Should sufficient information be known regarding a sequence alteration and confidence can be attributed to this being specific to a PBO line, then detection, identification and quantification can potentially be achieved. Genome editing and new mutagenesis techniques are umbrella terms, incorporating a plethora of approaches with diverse modes of action and resultant mutational changes. Generalisations regarding techniques and methods for detection for all PBO products are not appropriate, and each genome edited product may have to be assessed on a case-by-case basis. The application of modern molecular biology techniques, in isolation and by targeting just a single alteration, are unlikely to provide unequivocal evidence to the source of that variation, be that as a result of precision breeding or as a result of traditional processes. In specific instances, detection and identification may be technically possible, if enough additional information is available in order to prove that a DNA sequence or sequences are unique to a specific genome edited line (e.g., following certain types of Site-Directed Nucelase-3 (SDN-3) based approaches). The scope, gaps, and limitations associated with traceability of PBO products were examined, to identify current and future challenges. Alongside these, recommendations were made to provide the infrastructure for working towards a toolkit for the design, development and implementation of analytical methods for detection of PBO products. Recognition is given that fully effective methods for PBO detection have yet to be realised, so these recommendations have been made as a tool for progressing the current state-of-the-art for research into such methods. Recommendations for the following five main challenges were identified. Firstly, PBOs submitted for authorisation should be assessed on a case-by-case basis in terms of the extent, type and number of genetic changes, to make an informed decision on the likelihood of a molecular biology method being developed for unequivocal identification of that specific PBO. The second recommendation is that a specialist review be conducted, potentially informed by UK and EU governmental departments, to monitor those PBOs destined for the authorisation process, and actively assess the extent of the genetic variability and mutations, to make an informed decision on the type and complexity of detection methods that need to be developed. This could be further informed as part of the authorisation process and augmented via a publicly available register or database. Thirdly, further specialist research and development, allied with laboratory-based evidence, is required to evaluate the potential of using a weight of evidence approach for the design and development of detection methods for PBOs. This concept centres on using other indicators, aside from the single mutation of interest, to increase the likelihood of providing a unique signature or footprint. This includes consideration of the genetic background, flanking regions, off-target mutations, potential CRISPR/Cas activity, feasibility of heritable epigenetic and epitranscriptomic changes, as well as supplementary material from supplier, origin, pedigree and other documentation. Fourthly, additional work is recommended, evaluating the extent/type/nature of the genetic changes, and assessing the feasibility of applying threshold limits associated with these genetic changes to make any distinction on how they may have occurred. Such a probabilistic approach, supported with bioinformatics, to determine the likelihood of particular changes occurring through genome editing or traditional processes, could facilitate rapid classification and pragmatic labelling of products and organisms containing specific mutations more readily. Finally, several scientific publications on detection of genome edited products have been based on theoretical principles. It is recommended to further qualify these using evidenced based practical experimental work in the laboratory environment. Additional challenges and recommendations regarding the design, development and implementation of potential detection methods were also identified. Modern molecular biology-based techniques, inclusive of qPCR, dPCR, and NGS, in combination with appropriate bioinformatics pipelines, continue to offer the best analytical potential for developing methods for detecting PBOs. dPCR and NGS may offer the best technical potential, but qPCR remains the most practicable option as it is embedded in most analytical laboratories. Traditional screening approaches, similar to those for conventional transgenic GMOs, cannot easily be used for PBOs due to the deficit in common control elements incorporated into the host genome. However, some limited screening may be appropriate for PBOs as part of a triage system, should a priori information be known regarding the sequences of interest. The current deficit of suitable methods to detect and identify PBOs precludes accurate PBO quantification. Development of suitable reference materials to aid in the traceability of PBOs remains an issue, particularly for those PBOs which house on- and off-target mutations which can segregate. Off-target mutations may provide an additional tool to augment methods for detection, but unless these exhibit complete genetic linkage to the sequence of interest, these can also segregate out in resulting generations. Further research should be conducted regarding the likelihood of multiple mutations segregating out in a PBO, to help inform the development of appropriate PBO reference materials, as well as the potential of using off-target mutations as an additional tool for PBO traceability. Whilst recognising the technical challenges of developing and maintaining pan-genomic databases, this report recommends that the UK continues to consider development of such a resource, either as a UK centric version, or ideally through engagement in parallel EU and international activities to better achieve harmonisation and shared responsibilities. Such databases would be an invaluable resource in the design of reliable detection methods, as well as for confirming that a mutation is as a result of genome editing. PBOs and their products show great potential within the agri-food sector, necessitating a science-based analytical framework to support UK legislation, business and consumers. Differentiating between PBOs generated through genome editing compared to organisms which exhibit the same mutational change through traditional processes remains analytically challenging, but a broad set of diagnostic technologies (e.g., qPCR, NGS, dPCR) coupled with pan-genomic databases and bioinformatics approaches may help contribute to filling this analytical gap, and support the safety, transparency, proportionality, traceability and consumer confidence associated with the UK food chain.