Fw

dsDNA

ssDNA

FIGURE 8-1 Substrates required for DNA synthesis, (aj The general structure of the 2'-deoxynudeoside triphosphates. The positions of the o , p- and 7-phosphates are labeled- (b) The struc ture of a generalized pnmectemptate junction The shorter pnmer strand is completely annealed to the longer DNA strand and must have a free 3'OH adiacent to a ssDNA region of the template The longer DNA strand includes a region annealed to the primer and an adjacent ssDNA region that acts as the template for new DNA synthesis. New DNA synthesis extends the 3" end of the primer.

DNA Is Synthesized by Extending the 3' End of the Primer

The chemistry of DNA synthesis requires that the new chain grows by extending the 3' end of the primer (Figure 8-2). Indeed, this is a universal feature of thr synthesis of both RNA and DNA. The phosphndi-ester bond is formed in an SN2 reaction in which the hydroxyl group at the 3' end of the primer strand attacks the a-phosphoryl group of the incoming nucleoside triphosphate. The leaving group for the reaction is pyrophosphale, which arises from the release of the p- and y-phosphates of the nucleotide substrate.

The template strand direcls which of the four nucleoside triphosphates is added. The nucleoside triphosphate that base-pairs with the template strand is highly favored for addition to the primer strand. Recall that the two strands of the double helix have an antiparallel orientation. This arrangement means that the template strand for DNA synthesis has the opposite orientation of the growing DNA strand.

i _ _ pyrophosphatase

FIGURE 8-2 Diagram of the mechanism of DNA synthesis. DNA synthesis is initiated by the nLdeophiltc attack of the « phosphate of the incoming dNTP This results in the extension of the incoming 3' end of the primer by one nucleotide and the release of one molecule of pyrophosphate Pyrophosphatase rapidly hydrolyzes the pyrophosphate into two phosphate molecules.

primer template

Hydrolysis of Pyrophosphate Is the Driving Force for DNA Synthesis

The addition of a nucleotide to a growing polynucleotide chain of length n is indicated by the following reaction:

primer template i _ _ pyrophosphatase

FIGURE 8-2 Diagram of the mechanism of DNA synthesis. DNA synthesis is initiated by the nLdeophiltc attack of the « phosphate of the incoming dNTP This results in the extension of the incoming 3' end of the primer by one nucleotide and the release of one molecule of pyrophosphate Pyrophosphatase rapidly hydrolyzes the pyrophosphate into two phosphate molecules.

But the fren energy for this reaction is rather small (AC = -3.5 kcal/mole). What then is the driving force for the polymerization of nucleotides into DNA? Additional free energy is provided by the rapid hydrolysis of the pyrophosphate into two phosphate groups by an enzyme known as pyrophosphatase:

The net result nf nucleotide addition and pyrophosphate hydrolysis is the breaking of two high-eneigy phosphate bonds. Therefore, DNA synthesis is a coupled process, with an overall reaction of:

This is a highly favorable reaction with a AG of -7 kcal/mole which corresponds lo an equilibrium constant (K,.q) of about 10s. Such a high K^ means that the DNA synthesis reaction is effectively irreversible.

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