Unwinding Of DNA Double Helix Research Articles

Determination of the Thermodynamic Parameters of DNA Double Helix Unwinding with the Help of Mechanical Methods.

For the first time, rupture event scanning (REVS) procedure based on quartz crystal microbalance (QCM) and involving only mechanical action was used to determine the height of the energy barrier for dsDNA unwinding. Melting point was determined with the help of this procedure. To determine the thermodynamic parameters including enthalpy, DNA denaturation was represented as a unimolecular process. This allowed us to recover the energy profiles from the experimental data obtained by force measurements at different scanning times (reaction times) for different temperatures. The thus obtained results were compared with the data obtained with the help of another mechanical method, namely, atomic force microscopy. The mechanism of DNA unwinding in QCM-based experiments through the unzipping mode, as proposed by us in previous works, was confirmed. Thus, we demonstrated that REVS procedure may be used to assess the thermodynamic parameters of dsDNA unwinding.

Effect of the relief on the measurement of bond rupture force with the help of AFM: the dynamics of interaction and optimization of the procedure

Measurement of the forces of unwinding of DNA double helix was conducted.

In situ electrochemical and AFM study of thalidomide–DNA interaction

The interaction of thalidomide (TD) with double-stranded DNA (dsDNA) was studied using atomic force microscopy (AFM) at highly oriented pyrolytic graphite (HOPG), differential pulse voltammetry (DPV) at glassy carbon electrodes (GCE), UV–Vis and electrophoresis. After incubation of dsDNA with different concentrations of TD, the AFM images show the formation of thin and incomplete TD–DNA network films with a number of embedded molecular aggregates and regions of uncovered HOPG. Both the TD–dsDNA aggregates and network thickness directly depended on the TD concentration and incubation time. The voltammetric data also showed that the modifications caused by TD to the DNA double helical structure are time-dependent. In agreement with AFM, DPV, UV–Vis and electrophoresis results, a model is proposed for the TD–DNA interaction, considering that TD intercalates into the dsDNA, causing defects in the dsDNA secondary structure and DNA double helix unwinding. Moreover, both AFM and DPV show that condensation is caused to DNA by TD and occurs until 24 h of incubation, as well as DNA oxidative damage, detected electrochemically by the appearance of the 8-oxoGua and/or 2,8 oxoAde oxidation peak.

DNA Double Helix Unwinding Triggers Transcription Block-Dependent Apoptosis: A Semiquantitative Probe of the Response of ATM, RNAPII, and p53 to Two DNA Intercalators

We have previously shown the binding modes of two DNA interacting analogues (1)a {3-(4-methyl-piperazin)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} and (3)a {3-(3-dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} with the DNA double helix. In this study, we have determined the notably different DNA damage signal pathway elicited by (1)a and (3)a due to the different extents to which they unwind the DNA double helix. First, we have identified that ataxia-telangiectasia-mutated (ATM) protein kinase can respond to DNA double helix unwinding caused by both (1)a and (3)a. In addition, the amount of ATM activation is consistent with the degree to which the DNA double helix was unwound. Consequently, we used (1)a and (3)a to semiquantitatively probe the response of RNA polymerase II (RNAPII) and p53 toward DNA double helix unwinding in vivo. By means of flow cytometry, immunocytochemistry, ChIP, quantitative real-time polymerase chain reaction, and Western blot analyses, we measured the level of p53 and RNAPII phosphorylation, in addition to the dynamics of the RNAPII distribution along the c-Myc gene. These results provided novel evidence for the impact of subtle DNA structural changes on the activity of RNAPII and p53. Moreover, DNA double helix conformational damage-dependent apoptosis was studied for the first time. These results indicated that (1)a can induce transcriptional blockage following a shift of the unphosphorylated IIa form of RNAPII to the phosphorylated IIo form, while (3)a is unable to induce the same effect. Subsequently, p53 accumulation and phosphorylation events occur that lead to apoptosis in the case of (1)a exposure. This suggests that the transcriptional blockage is also correlated to the degree of double helix unwinding. Furthermore, we found that the degree of DNA conformational damage determines whether or not apoptosis occurs through transcriptional blockage. Under our experimental conditions, ATM does not participate in the downstream events even when it has been activated. Thus, p53-mediated apoptosis may be independently triggered by transcriptional blockage.

Role of unique basic residues of human pancreatic ribonuclease in its catalysis and structural stability

Human pancreatic ribonuclease (HPR) and bovine RNase A belong to the RNase A superfamily and possess similar key structural and catalytic residues. Compared to RNase A, HPR has six extra non-catalytic basic residues and high double-stranded RNA (dsRNA) cleavage activity. We mutated four of these basic residues, K6, R32, K62, and K74 to alanine and characterized the variants for function and stability. Only the variant K74A had an altered secondary structure. Whereas R32A and K62A had full catalytic activity, the mutants K6A and K74A had reduced activity on both ssRNA and dsRNA. The mutations of K62 and K74 resulted in reduction in protein stability and DNA double helix unwinding activity of HPR; while substitutions of K6 and R32 did not affect either the stability or helix unwinding activity. The reduced catalytic and DNA melting activities of K74A mutant appear to be an outcome of its altered secondary structure. The basic residues studied here, appear to contribute to the overall stability, folding, and general catalytic activity of HPR.

Structural Basis of Transcription: Nucleotide Selection by Rotation in the RNA Polymerase II Active Center

Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 Å.