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6. Definere begreberne proofereading og processivitet og angive i hvilen grad de forskellige DNA-polymeraser har disse egenskaber.

Proofreading
Stryer, s.752-3
Devlin, s. 164-5
Stryer, s.750 - fig. 27.11

DNA polymerases help ensure the accuracy of the replication process in two ways:

Initial selection - based on the fit of the incoming nucleotide into the enzymes active site. Only an incoming nucleotide that makes the correct hydrogen bonds with the nucleotide on the template strand can be added to the growing chain. The error rate is 10^-4

Proofreading - the proofreading mechanism relies on the increased probability that the end of the growing strand with an incorrectly incorporated nucleotide will leave the polymerase site and transiently move to the exonuclease site. This site has a 3´-5´endonucleatic activity. This means that it removes the last few incorporated bases on the 3´-end, including the wrongly incorporated base.

In other words, a primer with a properly base paired nucleotide is a good substrate for addition of the next nucleotide, and a poor substrate for the 3´-5´ endonuclease. On the other hand, a primer with a mismatched terminal nucleotide is a bad substrate for addition of the following nucleotides, and a good substrate for the 3´-5´endonuclease.

The result of proofreading is removal of virtually all mismatched nucleotides before the next nucleotide is added. It also leads to removal of some properly incorporated nucleotides.

DNA polymerases did not evolve ever-increasing accuracy of proofreading, probably because of the following reasons:

• extremely accurate proofreading is energetically costly

• it the process, a lot of correctly incorporated bases will be removed

• DNA repair systems found in the cell deal with residual errors

• a certain degree of mutations produces a population with some degree of genetical variation, which are better able to survive in changing conditions 

Processivity
Devlin, s.172, fig.4.10
Stryer, s.762

Processivity - the ability of an enzyme to catalyze many consecutive reactions without releasing its substrate.

The processivity unit of DNA polymerase III is a protein, called sliding clamp. They hold the DNA polymerase III in contact with the growing DNA chain. It is formed as a star-shaped ring. In the center of the ring there is a hole that can readily accommodate a duplex DNA molecule, yet leaving enough space between the DNA and the enzyme to allow rapid sliding and turning during replication.

A clamp-loading protein binds to DNA and assembles the sliding clamp. DNA polymerase binds in the place of the clamp loader and becomes processive.

A high processivity means also a fast addition of nucleotides, since the DNA is not released and caught again a lot of times.

DNA polymerase I has a endonuclease 3´-5´proofreading system, a relatively high processivity, (releases the DNA after adding 20 nucleotides) and a relatively fast synthesis (10 nucleotides/s).  

DNA polymerase III has also a endonuclease 3´-5´proofreading system, even higher processivity (doesn’t let go of DNA until the entire replication is finished) and an extremely fast synthesis (1000 nucleotides/s).

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