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We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the proliferative activity and neuroprotective effect of a newly identified ligustrazine derivative (4-((3,5,6-trimethylpyrazine-2-yl)methoxyl)-3-methox-ybenzoic acid-3,5,6- trimethylpyrazin- 2-methyl ester, T-VA) and the possible mechanism related to vascular endothelial growth factor (VEGF) in cerebral ischemic injury. Methods: The pharmacological activity of T-VA was evaluated using MTT ((3-(4,5-dimethylthiazolyl2- yl)-2,5-diphenyltetrazolium bromide)) assay, while cellular morphology was observed with hematoxylin and eosin (HE) staining. Chick chorioallantoic membrane (CAM) model, immuno-histochemical analysis, and enzyme-linked immunosorbent assay (ELISA) were used to determine the expression of VEGF. Middle cerebral artery occlusion (MCAO) model was used to investigate both VEGF expression and the survival rate after treatment with T-VA. Results: T-VA promoted neuron activity, and the doses of 15 and 30 µM showed more significant effect (p < 0.05). The viability of PC12 cells increased significantly in T-VA (30 and 60 µM) groups (p < 0.05) and increased in a dose-dependent manner. Immunohistochemical analysis showed stimulated VEGF expression, and CAM model results showed that T-VA (20 mg/egg) significantly promoted micro- angiogenesis (p < 0.01). Moreover, in MCAO model, the survival rate of T-VA (60 mg/kg) group reached 86.7 % while for the ischemia group it was 60.0 %. In addition, ELISA results showed that T-VA promoted the expression of VEGF (p < 0.05). Conclusion: These findings indicate that T-VA helps to prevent ischemic injury by increasing VEGF expression. [ABSTRACT FROM AUTHOR]

Bacteriophage RB69 DNA polymerase (RB69 pol) has served as a model for investigating how B family polymerases achieve a high level of fidelity during DNA replication. We report here the structure of an RB69 pol ternary complex at 1.8 Å resolution, extending the resolution from our previously reported structure at 2.6 Å [Franklin, M. C., et al. (2001) Cell 105, 657-667]. In the structure presented here, a network of five highly ordered, buried water molecules can be seen to interact with the N3 and O2 atoms in the minor groove of the DNA duplex. This structure reveals how the formation of the closed ternary complex eliminates two ordered water molecules, which are responsible for a kink in helix P in the apo structure. In addition, three pairs of polar-nonpolar interactions have been observed between (i) the Cα hydrogen of G568 and the N3 atom of the dG templating base, (ii) the O5' and C5 atoms of the incoming dCTP, and (iii) the OH group of S565 and the aromatic face of the dG templating base. These interactions are optimized in the dehydrated environment that envelops Watson-Crick nascent base pairs and serve to enhance base selectivity in wild-type RB69 pol.