Polymerases, specificity toward structure

It may be concluded that polymerases possess a rather broad specificity towards the structure of the monosaccharide residues that participate in formation of a new glycosidic linkage. Chemical synthesis of polyprenyl... 

There are, however, minor differences on the enzymes surfaces caused by amino acid insertions and deletions. These differences are most likely responsible for conferring specificity toward the interaction with factors specific for Pol I, II, and III. In addition to the 12 subunits that are either identical or homologous, Pol I contains two specific subunits, A34.5 and A49, and Pol III contains a subcomplex of three specific subunits, called C82, C34, and C31, in yeast. The location of the two Pol I—specific subunits has been determined by electron microscopy and immunolabeling (Bischler et al., 2002). The Pol I subunit A49 binds to the top of the clamp, and subunit A34.5 is located near the jaws. The location of the specific C82/C34/C31 complex of Pol III can be inferred from subunit-subunit interaction studies (Ferri et al., 2000 Flores et al., 1999). These studies indicate that the specific subcomplex is located between the largest polymerase subunit and the Rpb4/7 complex counterpart C17/C25. The Cll subunit of Pol III contains a C-terminal domain that apparently corresponds structurally and functionally to domain III of TFIIS (Chedin et al., 1998 Kettenberger et al., 2003), which inserts into the polymerase pore. Thus, in Pol III, the RNA cleavage stimulatory activity is incorporated into a polymerase subunit, in contrast to Pol II, where it is provided by the additional factor TFIIS. 

Probably even more important for wide, preparative use of the method is the fact that the specificity of the Salmonella O-antigen polymerases towards the structure of the trisaccharide unit was found to be not so strict as might be expected 29). A number... 

These studies demonstrated that (1) even a relatively minor chemical modification e.g., introduction of a 5-mercapto group into the cytosine base of 1 out of 100 nucleotide units) can convert a functional DNA or RNA template into a potent, competitive inhibitory analogue (antitemplate) which will bind to the template site of a polymerase either reversibly or irreversibly, and (2) even such antitemplates that are not made to be specific structural analogues of a natural template e.g., MPC) can differentiate between various polymerases. However, there are already some indications that antitemplates more closely related to the natural template of a given polymerase are more effective inhibitors of the latter, and it is expected that modified nucleic acids of viruses and tumors will show even much greater selectivities toward the corresponding reverse transcriptases in the presence of their endogenous templates. 

As a salutary post script to the discussion that has been presented above, a final example of innovation is presented, an example which has caused a profound change in direction in the search of anti-AIDS drugs. Until a little over a year ago, all research into inhibitors of reverse transcriptase (RT, see above) relied upon mimicry of the nucleoside substrates in the polymerase reaction. Here is an enzyme for which the reaction pathway is known only in bald outline and for which there is no structural information. Janssen s approach was masterly [84]. A library of 600 molecules, each prototypes of different chemical series and without activity in standard pharmacological assays, were screened for anti-HIV activity in vitro. It was discovered that (45) had modest but specific anti-HIV activity and lead optimisation eventually uncovered (46) and (47) as representatives of the TIBO series. It was subsequently shown that these compounds act as non-competitive inhibitors of RT, an indication that even if structural data were available, they would be useless in developing the series towards a drug candidate. As if by coincidence, several... 

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