DNA polymerase active sites

Where nucleic acids are concerned, the enhanced hydrophobicity of abiotic polyfluorinated aromatic bases (e.g., tetrafluorobenzene or tetrafluoroindole deoxyribose derivatives) was exploited as an alternative to natural hydrogen bonding to achieve selective and stable nucleic acid base pairing in duplex DNA [85], The DNA replication was examined using polyfluorinated-nucleotide analogs as substrates. A DNA polymerase active site was able to process the polyfluorinated base pairs more effectively than the analogous hydrocarbon pairs, demonstrating hydrophobic selectivity of polyfluorinated bases for other polyfluorinated bases [86]. 

J.S. Lai, E.T. Kool, Fluorous base-pairing effects in a DNA polymerase active site, Chem. Europ. J. 11 (2005) 2966-2971. 

Figure 3 Kinetic steps during DNA synthesis favor incorporation of correct dNTPs. Most often the DNA polymerase selects the correct dNTP that forms a correct Watson-Crick base pair with the template strand (pathway 1, left). The chemistry of correct dNTP incorporation is rapid, and it allows the polymerase to proceed rapidly to incorporate subsequent dNTPs. The chemistry of incorporating an incorrect dNTP is slow (pathway 2, right), and subsequent elongation of the mispaired 3 terminus is also slow. These two kinetic barriers provide time for the primed template to switch into the proofreading 3 -5 exonuclease active site, where removal of the mispaired 3 terminus is rapid. The excised and fully base paired primed site then switches back to the DNA polymerase active site (dashed arrow).

There are at least three types of nucleoside inhibitois behavior at the DNA polymerase active site ... 

Nevirapine binds directly to reverse transcriptase (RT) and blocks the RNA-dependent and DNA-dependent DNA polymerase activities by causing a disruption of the enzyme s catalytic site. The activity of nevirapine does not compete with P.1118... 

Stereoview of the polymerase active site of HIV-1 RT [38]. The amino acid residues that compose the putative dNTP-binding site, including the three catalytically essential aspartic acids, are shown with side chains. The double-stranded nucleic acid is shown with the atomic model in the HIV-1 RT/DNA/Fab complex. The dNTP-binding site consists of structural elements from both protein and nucleic acid. The precise composition, position, and conformation of the template-primer can affect the recognition of... 

A series of natural products, i.e., trihydroxyquinolone compounds isolated from Red Sea marine organisms, were reported to inhibit the DNA polymerase activity of HIV-1 RT [124,125]. This type of inhibitor appears to have a mechanism of inhibition that is different from either the NRTI inhibition mechanism or the NNRTI inhibition mechanism. The inhibition is reversible and noncompetitive with respect to both dNTP and template-primer [125]. This result indicates that there are other potential binding sites for inhibitors of HIV-1 RT. 

MECHANISM FIGURE 26-1 Transcription by RNA polymerase in E. coli. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound, (a) About 17 bp are unwound at any given time. RNA polymerase and the bound transcription bubble move from left to right along the DNA as shown facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. Red arrows show the direction in which the DNA must rotate to permit this process. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand extruded. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble, as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new nucleoside triphosphates (NTPs) to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation. 

Exonuclease activities, proofreading, and editing. DNA polymerase I not only catalyzes the growth of DNA chains at the 3 end of a primer strand but also, at about a 10-fold slower rate, the hydrolytic removal of nucleotides from the 3 end (31- 5 exonuclease activity). The same enzyme also catalyzes hydrolytic removal of nucleotides from the 5 end of DNA chains. This latter 5 - 3 exonuclease activity, the DNA polymerase activity, and the 3 -5 exonuclease activity all arise from separate active sites in the protein. DNA polymerases II and III do not catalyze... 

Nevirapine is a member of the dipyridodiazepinone class of chemicals and is a nonnucleoside reverse transcriptase inhibitor that induces a conformational change in HIV-1 reverse transcriptase. Although the conformational change is at a distance from its active site, it disrupts its catalytic activity. It blocks both RNA-dependent and DNA-dependent DNA polymerase activity but does not affect the activity of the template or nucleoside triphosphate. Nevirapine does not inhibit HIV-2 reverse transcriptase or human DNA polymerases a, (3 or y. The resistance to the drug results from site-directed mutagenesis at codons 103 or 181, and also at 100, 106, 108, 188 and 190 of viral reverse transcriptase. The development of resistance to one nonnucleoside reverse transcriptase implies that HIV will also be resistant to the rest of the drugs in this class. 

The initial event in the nucleotide incorporation cycle is binding of the double-stranded p/t DNA to the polymerase to form the E p/t complex (Step 1 Fig. 1). Many structures now exist, in which DNA is bound at the polymerase active site. 

The fidelity of polymerization depends on the ability of the polymerase active site to discriminate complementary from non-complementary incoming nucleotide triphosphates as well as to extend matched from unmatched primer termini. Taq DNA polymerase does not contain a 3 -5 exonudease or proofreading activity. This makes it advantageous for studying the intrinsic fidelity of the polymerase active site for discrimination between complementary versus non-complementary base pairing. 

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