Product-template duplex

Clearly, product dissociation by this scheme, step 3, will limit turnover, as the product-template duplex will be stabilized over the substrate-template ternary complex. Maximizing AAG in step 1 is required to enhance fidelity, but this increased affinity is generally present in the product duplex. Obviously if the binding affinity could be a tuned variable, high substrate affinity for ligation to maximize accuracy and reduced product affinity to enhance catalytic efficiency, product inhibition could be avoided. 

In Nature s genomes, repheation fidelity is primarily a kinetic function of the mechanistically complex DNA polymerases [43]. These enzymes employ activated phosphoric acid anhydrides leading to a final product-template duplex. The third step, dissociation of the product strand from the template, requires an additional input of chemical energy and a separate set of protein catalysts. Therefore each step in DNA replication requires a critical and complex set of translation products to ensure the production of a single copy of the template, and for autonomous growth, the template must be of sufficient size to encode all of the required catalysts for the process. The notion that these templates must encode catalysts, or possibly even the templates themselves serve as catalysts for more than one reaction, becomes an important feature for the development of template autonomy. 

The condensation reaction yields an imine 88 with the appropriate set of hydrogen bond donor/acceptor groups to template its own formation via a ternary complex (involving the product and the two reactants). Closer inspection to this reaction has revealed that the tertiary complex is actually more stable (in some of the reaction studied) than the duplex formed between the template and the product. Consequently, once the templation has taken place, the duplex is separated and both the product and original template are ready to accelerate the reaction of the two reactants. Since the number of templates has now doubled, the enhancement of the reaction could in principle follow an exponential rate. 

Detailed structures of several DNA polymerases are available, including those with double- or single-stranded DNA bound.31-35 Site-directed mutagenesis (Chapter 14) has been used to identify residues that are in the active sites for polymerization and exonuclease activity.36,37 Mutants that are defective in the exonuclease activity are used for the production of stable complexes with single-stranded DNA or single-stranded 3 ends of duplexes bound in the exonuclease site.31,33 2, 3 -Dideoxynucleotides (ddNTPs) that lack the nucleophilic 3 -OH have been used to make templates that are inert so that productive ternary enzyme DNA ddNTP complexes may be observed.34,35,38... 

Figure 7.1 Schematic representation of PCR N0 copies of duplex template DNA are subjected to n cycles of PCR. During each cycle, duplex DNA is denatured by heating, allowing primers (arrows) to anneal to the targeted sequence (hatched square). In the presence of DNA polymerase and dNTPs, the primers are extended. The desired blunt-ended duplex product (thick bars with arrows) does not appear until after the third cycle, and accumulates exponentially during subsequent cycles. After n cycles of PCR, N0 (1 + Y)""1 copies of duplex product are present. [Reprinted with permission from Cha and Thilly, PCR Methods Appl 3 S18 (1993).]...

When replication occurs, the two strands of the Watson-Crick double helix must separate so that each can serve as a template for the synthesis of its complement. Since the two strands are complementary to one another, each bears a definite sequence relationship to the other. When one strand acts as a template, it directs the synthesis of its complement. The product of the synthesis directed by each template strand is therefore a duplex molecule that is identical to the starting duplex. The process is accurate because of the specificity of base pairing and because the protein apparatus that catalyzes the replication can remove mismatched bases. 

Figure 4.8 DNA machine based on strand displacement amplification. The track consists of a primer binding sequence (black, I), a nicking recognition sequence (green, II), and a template for the product DNA (blue, IE). (1) Binding of the primer starts replication of the entire track by a DNA polymerase. (2) The fully replicated duplex is nicked on the newly formed strand by the nicking enzyme, regenerating the primer site...

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