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A single-nucleotide polymorphism detection assay using PCR with quenching probes (QP-PCR) was developed for the rapid detection of antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). The mutations examined were in the HHV-6 U69 gene, and were single-base mutations in sequences known to be associated with ganciclovir (GCV) resistance in HCMV. We previously confirmed that they conferred GCV resistance to recombinant baculoviruses (Nakano et al., J. Virol. Methods 161:223-230, 2009). Six characterized mutations, including a previously reported one that encodes a GCV-sensitive kinase-activity mutant (Isegawa et al., J. Clin. Virol. 44:15-19, 2009), were used. The six mutations were separated into three groups based on their location in the U69 protein, and detected by the hybridization of three probes. We developed and validated a set of assays for these mutations using PCR followed by differential melting of a fluorescently labeled oligo probe, on a Roche Light Cycler platform. Nucleobase quenching was used to detect the hybridized probe. The optimized assay could distinguish the different mutants, and easily detected mutants representing 30% of the DNA in a mixed sample. This QP-PCR assay permitted the rapid (1.5 h), objective, and reproducible detection of drug-resistant mutations of HHV-6., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In this chapter, we discuss a new method for quantifying DNA-protein interactions. A single double-stranded DNA (dsDNA) molecule is stretched beyond its contour length, causing the base pairs to break while increasing the length from that of dsDNA to that of ssDNA. When applied in a solution containing DNA binding ligands, this method of force-induced DNA melting can be used to quantify the free energy of ligand binding, including the free energy of protein binding. The dependence of melting force on protein concentration is used to obtain the equilibrium binding constant of the ligand to DNA. We have applied this method to a well-studied DNA-binding protein, bacteriophage T4 gene 32 protein (gp32), and have obtained binding constants for the protein to single-stranded DNA (ssDNA) under a wide range of solution conditions. Our analysis of measurements conducted at several salt concentrations near physiological conditions indicates that a salt-dependent conformational change regulates DNA binding by gp32.

Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged., (Copyright 2000 Academic Press.)

The helix-destabilizing properties of a single-stranded DNA-binding protein, LEF-3, of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Partial duplexes of DNA containing single-stranded (ss) tails of different sizes and orientations were used as substrates for assay of the unwinding ability of LEF-3. Upon noncooperative binding to ssDNA, LEF-3 was capable of unwinding the duplexes with 5' ss tails. However, it did not cause melting of the duplexes containing 3' ss tails, even at oversaturation of ssDNA adjacent to the duplexes. Upon cooperative binding to long ss tails, LEF-3 also produced the polar melting effect; it unwound the duplexes with long 5' ss tails, but not those with long 3' ss tails. These data suggest that LEF-3 has a preferential direction for entry into duplex DNA, namely 5' to 3' with respect to the bound DNA strand. In agreement with its polarity, LEF-3 efficiently melted the primer-template complexes which serve as substrates for DNA polymerases. However, the formation of a complex with viral DNA polymerase before addition of LEF-3 protected the primer-templates from the destabilization effect of LEF-3. Although the destabilization effect of LEF-3 was highly sensitive to monovalent and divalent salts, the protein was capable of melting DNA duplexes in a polar manner at physiological conditions, i.e., 30 degrees C in 0.15 M NaCl. Therefore, the polar destabilization effect of LEF-3 seems to be physiologically important and may be connected, in particular, with the polar action of viral helicase holoenzyme during baculovirus replication., (Copyright 2000 Academic Press.)

The products of interaction of acridine orange (AO) with single-stranded (ss) nucleic acids are precipitates which exhibit red luminescence. Titration of rRNA or thymus DNA with AO results in formation of such products suggesting that the dye, per se, denatures double-stranded (ds) sections of these biopolymers. This transition, measured as the increase of red luminescence, a concomitant decrease of green fluorescence, and followed by an increase of light scatter of the AO-nucleic acid complexes, is cooperative and at 0.15 N NaCl occurs at 4-20 and 10-50 microM range of AO concentration for rRNA and DNA, respectively. The changes in stainability of nucleic acids in situ, in permealized cells, occur at higher AO concentration. Thus, the transition of RNA in situ is biphasic and seen at 20-120 microM AO. In the presence of EDTA, however, the change is monophasic and shifted to the 10-30 microM range of AO concentration. The change in stainability of DNA also shows two phases: one at 30-60 microM and another at 70-120 microM of AO. Extraction of basic proteins with 0.08 N HCl shifts the transition of DNA to the 30-60 microM AO concentration and makes it monophasic. The observed differences in denaturability of RNA vs DNA explain the specificity of AO in differential staining of these bipolymers in histochemical reactions. In living cells the products of interaction of AO with nucleic acids are detected by electron microscopy. In the cytoplasm of interphase cells the formation of dense precipitates within ribosomes and polysomes, simultaneous with a specific retraction of ribosome-polysome complexes from the periphery of the cell to the nucleus is evident. The latter suggests higher order organization of these particles involving their association with each other or with the nucleus via polyanionic macromolecules which collapse upon binding with AO. The DNA in heterochromatin is more sensitive to AO-induced denaturation, as evidenced by the fact that the dense complexes are formed preferentially in the regions of condensed chromatin of the interphase nucleus, or in metaphase chromosomes.