Modulating Polymerase Activity Through Light-Oxygen-Voltage Domain Insertion.

Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase.

Chemical Evolution of Natural Product Structure.

Natural products are the result of Nature's exploration of biologically relevant chemical space through evolution and an invaluable source of bioactive small molecules for chemical biology and medicinal chemistry. Novel concepts for the discovery of new bioactive compound classes based on natural product structure may enable exploration of wider biologically relevant chemical space. The pseudo-natural product concept merges the relevance of natural product structure with efficient exploration of chemical space by means of fragment-based compound development to inspire the discovery of new bioactive chemical matter through combination of natural product fragments in unprecedented arrangements.

Pseudo Natural Products-Chemical Evolution of Natural Product Structure.

Pseudo-natural products (PNPs) combine natural product (NP) fragments in novel arrangements not accessible by current biosynthesis pathways. As such they can be regarded as non-biogenic fusions of NP-derived fragments. They inherit key biological characteristics of the guiding natural product, such as chemical and physiological properties, yet define small molecule chemotypes with unprecedented or unexpected bioactivity.

Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase.

Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity.

LOV Domains in the Design of Photoresponsive Enzymes.

In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occurring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest.

The double mutation L109M and R448M of HIV-1 reverse transcriptase decreases fidelity of DNA synthesis by promoting mismatch elongation.

Changes of Leu109 and Arg448 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have as yet not been associated with altered fitness. However, in a recent study, we described that the simultaneous substitution of L109 and R448 by methionine leads to an error-producing polymerase phenotype that is not observed for the isolated substitutions. The double mutant increased the error rate of DNA-dependent DNA synthesis 3.

Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2'-O-methyl-modified RNA.

T7 RNA polymerase is an important biocatalyst that is used in diverse biotechnological applications such as in vitro transcription or protein expression. The enzyme displays high substrate specificity which is payed by significant limitations regarding incorporation of synthetic nucleotide analogs. Of specific interest is enzymatic synthesis of 2'-O-methyl-modified RNA as these nucleic acids exhibit improved biochemical and pharmacokinetic properties that make them attractive for diagnostic and therapeutic purposes.

Exonucleolytic degradation of high-density labeled DNA studied by fluorescence correlation spectroscopy.

The exonucleolytic degradation of high-density labeled DNA by exonuclease III was monitored using two-color fluorescence correlation spectroscopy (FCS). One strand of the double stranded template DNA was labeled on either one or two base types and additionally at one end via a 5' Cy5 tagged primer. Exonucleolytic degradation was followed via the diffusion time, the brightness of the remaining DNA as well as the concentration of released labeled bases.

Directed evolution of an error-prone T7 DNA polymerase that attenuates viral replication.

Experimental evidence exists that RNA viruses replicate with extremely high mutation rates that result in significant genetic diversity. The diverse nature of viral populations allows rapid adaptation to dynamic environments, and evolution of resistances to vaccines as well as antiviral substances. For DNA viruses that replicate at much greater fidelities, as yet, neither diverse structures in the population nor their responses to increased mutation rates have been sufficiently described.

Activity-based selection of HIV-1 reverse transcriptase variants with decreased polymerization fidelity.

HIV-1 reverse transcriptase (HIV-1 RT) copies the RNA genome of HIV-1 into DNA, thereby committing errors at an exceptionally high frequency. Viral offspring evolve rapidly and consequently are capable of evading the immune response as well as antiviral treatment. However, error-prone viral replication could drive HIV close to extinction owing to an intolerable load of deleterious mutations.

Exonuclease III action on microarrays: observation of DNA degradation by fluorescence correlation spectroscopy.

DNA with all cytosines, thymines, or all pyrimidines of one strand substituted by fluorescently labeled analogs shows diminished solubility in aqueous media and a strong tendency to aggregation that hampers enzymatic downstream processing. In this study, immobilization of fully fluorescently labeled DNA on microarrays was shown to resolve the named problems and to enable successive DNA degradation by exonuclease III. Fluorescence correlation spectroscopy and single-molecule counting for monitoring the course of DNA hydrolysis in real time revealed the virtually processive degradation of labeled DNA that occurred at an average rate of approximately 4 nt/s.

Sequencing single DNA molecules in real time.

One's enough: The direct observation of a DNA-polymerase-based "sequencing engine" using single-molecule detection recently allowed single-molecule sequencing by synthesis in real time. Nucleotides with a fluorescent marker at the 5'-phosphate unit and zero-mode waveguides are crucial components of this approach, which at last promises low-cost genome-scale sequencing.

Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase.

The formation of inclusion bodies is a frequent consequence of high-level production of foreign protein in the cytoplasm of Escherichia coli. This phenomenon is also observed with bacteriophage T7 gene 5 protein, the phage-encoded subunit of T7 DNA polymerase, if expression is based on the T5 promoter/lac operator transcription-translation system present in a vector with ColE1 origin of replication. To avoid tedious procedures for recovering protein from insoluble aggregates, we studied the expression of T7 gene 5 protein using a series of E.

High-density labeling of DNA for single molecule sequencing.

Two unusual enzymatic activities are required for the realization of a single molecule sequencing: a polymerase for copying a deoxyribonuclease (DNA) target into complementary flurophore-labeled DNA, and an exonuclease for the successive hydrolysis of the completely dye-labeled DNA. Recently, we found that the wild-type Klenow fragment of Escherichia coli DNA polymerase I is well-suited for the synthesis of DNA in a reaction set-up that contains exclusively specific rhodamine-labeled analogs of the natural pyrimidine nucleotides (dCTP and dTTP). This protocol describes the procedure used for the preparation of DNA that is labeled at all pyrimidine bases of one strand, as well as an example of enzymatic downstream processing of the DNA product.

Optimal enzymes for single-molecule sequencing.

Much research effort has been made to realize a single-molecule sequencing. Central to this project are two enzymatic tasks that challenged several biochemists during the last years. They searched for polymerases for copying a DNA target into completely fluorophore-labeled DNA as well as for handling this new DNA derivative.