Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model.

Recombinational repair of double-stranded DNA gaps was investigated in Ustilago maydis. The experimental system was designed for analysis of repair of an autonomously replicating plasmid containing a cloned gene disabled by an internal deletion. It was discovered that crossing over rarely accompanied gap repair. The strong bias against crossing over was observed in three different genes regardless of gap size. These results indicate that gap repair in U. maydis is unlikely to proceed by the mechanism envisioned in the double-stranded break repair model of recombination, which was developed to account for recombination in Saccharomyces cerevisiae. Experiments aimed at exploring processing of DNA ends were performed to gain understanding of the mechanism responsible for the observed bias. A heterologous insert placed within a gap in the coding sequence of two different marker genes strongly inhibited repair if the DNA was cleaved at the promoter-proximal junction joining the insert and coding sequence but had little effect on repair if the DNA was cleaved at the promoter-distal junction. Gene conversion of plasmid restriction fragment length polymorphism markers engineered in sequences flanking both sides of a gap accompanied repair but was directionally biased. These results are interpreted to mean that the DNA ends flanking a gap are subject to different types of processing. A model featuring a single migrating D-loop is proposed to explain the bias in gap repair outcome based on the observed asymmetry in processing the DNA ends.

Sequence scanning: A method for rapid sequence acquisition from large-fragment DNA clones.

A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.

Synthesis of a thymidyl pentamer of deoxyribonucleic guanidine and binding studies with DNA homopolynucleotides.

Replacement of the phosphodiester linkages of the polyanion DNA with guanidine linkers provides the polycation deoxynucleic guanidine (DNG). The synthesis of pentameric thymidyl DNA is provided. This polycationic DNG species binds with unprecedented affinity and with base-pair specificity to negatively charged poly(dA) to provide both double and triple helices. The dramatic stability of these hybrid structures is shown by their denaturation temperatures (Tm). For example, the double helix of the pentameric thymidyl DNG and poly(dA) does not dissociate in boiling water (ionic strength = 0.12), whereas the Tm for pentameric thymidyl DNA associated with poly(dA) is approximately 13 degrees C (ionic strength = 0.12). The effect of ionic strength on Tm for DNG complexes with DNA shows an opposite correlation compared with double-stranded DNA and is much more dramatic than for double-stranded DNA.

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

The Structure of theCyprinid herpesvirus 3ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

In vivo gene expression: DNA electrotransfer

The use of electric pulses to deliver therapeutic molecules to tissues and organs in vivo is a rapidly growing field of research. Electrotransfer can be used to deliver a wide range of potentially therapeutic agents, including drugs, proteins, oligonucleotides, RNA and DNA. Optimization of this approach depends upon a number of parameters such as target organ accessibility, cell turnover, microelectrode design, electric pulsing protocols and the physiological response to the therapeutic agent. Many organs have been successfully transfected by electroporation, including skin, liver, skeletal and cardiac muscle, male and female germ cells, artery, gut, kidney, retinal ganglion cells, cornea, spinal cord, joint synovium and brain. Electrotransfer technology is relevant in a variety of research and clinical settings including cancer therapy, modulation of pathogenic immune reactions, delivery of therapeutic proteins and drugs, and the identification of drug targets by the modulation of normal gene expression. This, together with the capacity to deliver very large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer.

Higher-order CpG-DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single-stranded CpG-oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG-ODN capable of forming higher-order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell-activating factor, or simultaneous co-engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF-1, IL-6 and IL-12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self-derived hypomethylated double-stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co-engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9-induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies

Antibodies reactive with native double stranded DNA are characteristic of the chronic inflammatory disease systemic lupus erythematosus. Native DNA is however, a poor immunogen and the mechanism of anti-DNA antibody production is incompletely understood. Modification of DNA can increase its immunogenicity and in inflammatory disease states reactive oxygen species produced from phagocytic cells have been shown to thus modify DNA. In this study, monoclonal antibodies produced spontaneously by two mice strains with lupus-like disease were used in a competition ELISA to monitor changes to DNA induced by reactive oxygen species. Different procedures for reactive oxygen species generation were found to cause distinct and characteristic changes to DNA involving modifications of base residues, the sugar-phosphate backbone and the gross conformational structure of double-stranded DNA. In view of this, it may be possible to use these antibodies further to probe DNA and infer the source and nature of the reactive oxygen species it has been exposed to, particularly in vivo.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

RECQ5 promotes recombination and mutagenesis at targeted nicks through disruption of RAD51 filaments

Thesis (Ph.D.)--University of Washington, 2016-08

DNA damage induced by low energy electrons (LEEs)

Abstract : The major objective of our study is to investigate DNA damage induced by soft X-rays (1.5 keV) and low-energy electrons (˂ 30 eV) using a novel irradiation system created by Prof. Sanche’s group. Thin films of double-stranded DNA are deposited on either glass and tantalum substrates and irradiated under standard temperature and pressure surrounded by a N[subscript 2] environment. Base release (cytosine, thymine, adenine and guanine) and base modifications (8-oxo-7,8-dihydro -2’-deoxyguanosine, 5-hydroxymethyl-2’-deoxyuridine, 5-formyl-2’-deoxyuridine, 5,6-dihydrothymidine and 5,6-dihydro-2’-deoxy uridine) are analyzed and quantified by LC-MS/MS. Our results reveal larger damage yields in the sample deposited on tantalum than those on glass. This can be explained by an enhancement of damage due to low-energy electrons, which are emitted from the metal substrate. From a comparison of the yield of products, base release is the major type of damage especially for purine bases, which are 3-fold greater than base modifications. A proposed pathway leading to base release involves the formation of a transient negative ion (TNI) followed by dissociative electron attachment (DEA) at the N-g lycosidic bond. On the other hand, base modification products consist of two major types of chemical modifications, which include thymine methyl oxidation products that likely arises from DEA from the methyl group of thymine, and 5,6-dihydropyrimidine that can involve the initial addition of electrons, H atoms, or hydride ions to the 5,6-pyrimidine double bond.

The effect of the dibenzylbutyrolactolic lignan (-)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila melanogaster

The dibenzylbutyrolactolic lignan (-)-cubebin was isolated from dry seeds of Piper cubeba L (Piperaceae). (-)-Cubebin possesses anti-inflammatory, analgesic and antimicrobial activities. Doxorubicin (DXR) is a topoisomerase-interactive agent that may induce single- and double-strand breaks, intercalate into the DNA and generate oxygen free radicals. Here, we examine the mutagenicity and recombinogenicity of different concentrations of (-)-cubebin alone or in combination with DXR using standard (ST) and high bioactivation (HB) crosses of the wing Somatic Mutation And Recombination Test in Drosophila melanogaster. The results from both crosses were rather similar. (-)-Cubebin alone did not induce mutation or recombination. At lower concentrations, (-)-cubebin statistically reduced the frequencies of DXR-induced mutant spots. At higher concentrations, however, (-)-cubebin was found to potentiate the effects of DXR, leading to either an increase in the production of mutant spots or a reduction, due to toxicity. These results suggest that depending on the concentration, (-)-cubebin may interact with the enzymatic system that catalyzes the metabolic detoxification of DXR, inhibiting the activity of mitochondria! complex 1 and thereby scavenging free radicals. Recombination was found to be the major effect of the treatments with DXR alone. The combined treatments reduced DXR mutagenicity but did not affect DXR recombinogenicity. (C) 2011 Elsevier Ltd. All rights reserved.