Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytomegalovirus (CMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in CMV infection is well documented. Traditionally the virus has been propagated in fibroblasts, however this process may alter CMV's characteristics, thereby limiting the fibroblast model's utility as a research tool. In our efforts to develop a more accurate in vitro model of CMV/endothelial cell interaction, we have propagated a recent isolate (CMV VHL) through multiple passages in human umbilical vein endothelial cells (HUVE) and, collaterally in neonatal human dermal fibroblasts (NHDF). Infection of HUVE inoculated with either sub-strain of the virus was confirmed by CMV-specific in situ hybridization and by immunocytochemical staining for CMV antigens. Whereas infection of HUVE by substrain VHL/E (endothelial-raised) was accompanied by dramatic cytopathology resembling that observed clinically, the endothelial cytopathic potential of VHL/F (fibroblast-raised) was lost by its 20th passage in NHDF. Similarly, the ability of VHL/F to initiate sustained productive infection in HUVE was severely attenuated; plaque assay of culture supernatants and infected cell fractions, as well as virus-specific DNA polymerase assay of cell lysates, demonstrated progressive viral reproductive activity in VHL/E-inoculated HUVE, whereas VHL/F reproduction was barely detectable. Since properties of VHL/F bear strong resemblance to those of the fibroblast-raised AD169, these studies suggest that while the fibroblast adaptation process commonly employed in the propagation of CMV restricts the host range of the virus and attenuates its spectrum of cytopathic potential, endothelial-based propagation preserves the natural endothelial cytopathogenicity of the original isolate.
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PMID:Preservation of natural endothelial cytopathogenicity of cytomegalovirus by propagation in endothelial cells. 185 Feb 27

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.
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PMID:Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system. 394 76

The incorporation of m6dATP by T4 DNA polymerase has been investigated. Unlike Escherichia coli DNA polymerase I (Engel, J.D., and von Hippel, P.H. (1978) J. Biol. Chem, 253, 935-939), the T4 enzyme discriminates at the insertion step against the methylated triphosphate as compared to the normal substrate (dATP). The apparent Km values measured in two ways agree with the overall 7-fold discrimination measured in double label experiments. The apparent Vmax values measured for net DNA synthesis are the same, while those measured for nucleotide turnover show that the rate for m6dATP is 2-fold greater than for dATP itself. The T4 enzyme results are consistent with the generally held theory that fidelity at the insertion step of DNA polymerization is determined by the relative free energies of primer-enzyme-triphosphate ternary complexes formed by competing, alternative substrate dNTPs. These results are also consistent with the view that these free energies chiefly depend on formation of satisfactory hydrogen bonds between the bases of the template and triphosphate.
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PMID:N6-methyldeoxyadenosine 5'-triphosphate as a probe of the fidelity mechanisms of bacteriophage T4 DNA polymerase. 670 72

In the preceding paper (Latham, G. J., Bacheller, D. J., Pietroni, P. , and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31677-31684), we demonstrated that the T4 gp44/62-ATP clamp loader binds to the C-terminal face of the gp45 sliding clamp. Here we extend these results by exploring the structural relationship between the gp43 polymerase and the gp45 sliding clamp. Using fluorescence intensity and polarization techniques, as well as photo-cross-linking methods, we present evidence that gp43, like gp44/62, binds to the C-terminal face of gp45. In addition, we show that g43 binds to the gp45 clamp in two distinct interaction modes, depending on the presence or absence of template-primer DNA. When template-primer DNA is present, gp43 binds tightly to gp45 to form the highly processive DNA polymerase holoenzyme. Gp43 also binds to gp45 in the absence of template-primer DNA, but this interaction is more than 100 times weaker than gp43-gp45 binding on DNA. Specific interactions between gp43 and the C-terminal face of gp45 are maintained in both modes of binding. These results underscore the pivotal role of template-primer DNA in modulating the strength of protein-protein interactions during DNA synthesis and provide additional insight into the structural requirements of the replication process.
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PMID:Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. III. The Gp43 DNA polymerase binds to the same face of the sliding clamp as the clamp loader. 939 10