14,621 research outputs found
Reconstitution of recombination-associated DNA synthesis with human proteins.
The repair of DNA breaks by homologous recombination is a high-fidelity process, necessary for the maintenance of genome integrity. Thus, DNA synthesis associated with recombinational repair must be largely error-free. In this report, we show that human DNA polymerase delta (Ξ΄) is capable of robust DNA synthesis at RAD51-mediated recombination intermediates dependent on the processivity clamp PCNA. Translesion synthesis polymerase eta (Ξ·) also extends these substrates, albeit far less processively. The single-stranded DNA binding protein RPA facilitates recombination-mediated DNA synthesis by increasing the efficiency of primer utilization, preventing polymerase stalling at specific sequence contexts, and overcoming polymerase stalling caused by topological constraint allowing the transition to a migrating D-loop. Our results support a model whereby the high-fidelity replicative DNA polymerase Ξ΄ performs recombination-associated DNA synthesis, with translesion synthesis polymerases providing a supportive role as in normal replication
Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe
The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5β10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR
Control of DNA Strand Displacement Kinetics Using Toehold Exchange
DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices
Scalable, Time-Responsive, Digital, Energy-Efficient Molecular Circuits using DNA Strand Displacement
We propose a novel theoretical biomolecular design to implement any Boolean
circuit using the mechanism of DNA strand displacement. The design is scalable:
all species of DNA strands can in principle be mixed and prepared in a single
test tube, rather than requiring separate purification of each species, which
is a barrier to large-scale synthesis. The design is time-responsive: the
concentration of output species changes in response to the concentration of
input species, so that time-varying inputs may be continuously processed. The
design is digital: Boolean values of wires in the circuit are represented as
high or low concentrations of certain species, and we show how to construct a
single-input, single-output signal restoration gate that amplifies the
difference between high and low, which can be distributed to each wire in the
circuit to overcome signal degradation. This means we can achieve a digital
abstraction of the analog values of concentrations. Finally, the design is
energy-efficient: if input species are specified ideally (meaning absolutely 0
concentration of unwanted species), then output species converge to their ideal
concentrations at steady-state, and the system at steady-state is in (dynamic)
equilibrium, meaning that no energy is consumed by irreversible reactions until
the input again changes.
Drawbacks of our design include the following. If input is provided
non-ideally (small positive concentration of unwanted species), then energy
must be continually expended to maintain correct output concentrations even at
steady-state. In addition, our fuel species - those species that are
permanently consumed in irreversible reactions - are not "generic"; each gate
in the circuit is powered by its own specific type of fuel species. Hence
different circuits must be powered by different types of fuel. Finally, we
require input to be given according to the dual-rail convention, so that an
input of 0 is specified not only by the absence of a certain species, but by
the presence of another. That is, we do not construct a "true NOT gate" that
sets its output to high concentration if and only if its input's concentration
is low. It remains an open problem to design scalable, time-responsive,
digital, energy-efficient molecular circuits that additionally solve one of
these problems, or to prove that some subset of their resolutions are mutually
incompatible.Comment: version 2: the paper itself is unchanged from version 1, but the
arXiv software stripped some asterisk characters out of the abstract whose
purpose was to highlight words. These characters have been replaced with
underscores in version 2. The arXiv software also removed the second
paragraph of the abstract, which has been (attempted to be) re-inserted.
Also, although the secondary subject is "Soft Condensed Matter", this
classification was chosen by the arXiv moderators after submission, not
chosen by the authors. The authors consider this submission to be a
theoretical computer science paper
Double-strand break repair and homologous recombination in Schizosaccharomyces pombe
In recent years our understanding of double strand break repair and homologous recombination in Schizosaccharomyces pombe has increased significantly, and the identification of novel pathways and genes with homologues in higher eukaryotes has increased its value as a model organisms for double strand break repair. We will review the S. pombe literature on double strand break repair, mainly focussing on homologous recombination in mitotic cells
The Boltzmann factor, DNA melting, and Brownian ratchets: Topics in an introductory physics sequence for biology and premedical students
Three, interrelated biologically-relevant examples of biased random walks are
presented: (1) A model for DNA melting, modelled as DNA unzipping, which
provides a way to illustrate the role of the Boltzmann factor in a venue
well-known to biology and pre-medical students; (2) the activity of helicase
motor proteins in unzipping double-stranded DNA, for example, at the
replication fork, which is an example of a Brownian ratchet; (3) force
generation by actin polymerization, which is another Brownian ratchet, and for
which the force and actin-concentration dependence of the velocity of actin
polymerization is determined
Capturing the βomeβ : the expanding molecular toolbox for RNA and DNA library construction
All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application
Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.
The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 Β΅L reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment
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