Single-step intercalating dye strategies for DNA damage studies.

Many analytical protocols exist for the quantification of varied types of DNA damage, which span a range of complexity and sensitivity. As an alternative or companion to existing procedures, this article demonstrates the application of quantitative PCR (qPCR) and high-resolution DNA melting analysis (HRMA) to the detection and quantification of intramolecular DNA damage and/or strand breaks. These proven molecular biology methods are essentially single-step processes. When implemented with a third-generation saturating DNA dye, high sensitivity can be obtained. The experiments presented here demonstrate how DNA damage can inhibit amplification of the affected molecules. This corresponding decrease in the initial concentration of amplifiable DNA can be measured with qPCR. In addition, damage in the form of intramolecular dimerization and strand breaks alters the stored energy in the hydrogen bonds between the two strands in the dsDNA molecule. This significantly affects the thermal stability, which can be measured with extreme precision using HRMA. Simplified damage models were used in these experiments: UV-C irradiation to produce photoproducts, and restriction enzyme digestion to simulate double-strand breaks. The findings of this work, however, can be intuitively applied to the broad scope of DNA damage mechanisms.

Glass-composite prototyping for flow PCR with in situ DNA analysis.

In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 microl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 microl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (PhiX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.

Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry.

Human papillomaviruses (HPV) infect stratified epithelia and restrict expression of late capsid genes to highly differentiated cells. In order to begin to understand the processes regulating HPV 31 infection we examined the synthesis of the HPV 31 capsid proteins, L1 and L2, using heterologous expression systems. Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis. In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis. Through the use of chimeric proteins that fused fragments of wild type L1 to Green Fluorescent Protein (GFP) coding sequences, a short region was identified that was sufficient to inhibit high level synthesis and similar elements were detected in L2. One element was localized to the 3' end of the L1 gene while a series of elements were localized at the 3' end of the L2 coding sequences. These observations are most consistent with negative RNA regulatory elements controlling the levels of L1 and L2 synthesis that are distinct from those identified in HPV 16. Expression vectors for the codon modified HPV 31 capsid proteins were then transfected together with GFP reporter plasmids to generate HPV 31 pseudoviruses. Infection of cells with HPV 31 pseudoviruses in the presence of the inhibitors, chlorpromazine, nystatin or methyl-beta-cyclodextrin, demonstrated that HPV 31, like HPV 16, enters human and monkey cells through a clathrin-mediated pathway rather than through caveolae as previously reported. This suggests that high-risk HPV types may enter cells through common mechanisms.

Subcellular localization and integration activities of rous sarcoma virus reverse transcriptase.

Reverse transcriptases (RTs) alphabeta and beta from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT alphabeta and the beta subunit, when expressed independently, can be detected in the nucleus, whereas the separate alpha subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of beta, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT alphabeta with an oligodeoxynucleotide-based assay system and with a donor substrate containing the supF gene flanked by the viral long terminal repeats. Our data show that RSV RT alphabeta is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT alphabeta could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).