DNA Replication

DNA replication is a semi-conservative process. One strand serves as the template for the second strand. DNA replication is initiated at a region on a chromosome called an origin of replication. An enzyme called DNA Helicase binds to the origin and unwinds the DNA in both directions from the origin.

As the DNA is unwound, specific single stranded DNA binding proteins prevent the strands from reannealing. RNA primers are produced by primase, which bind to the DNA. The DNA polymerase then binds and begins to synthesize the DNA complementary to the parental strand.

DNA replication has two requirements that must be met:

1. A DNA template.
2. A free 3' -OH group.

Proteins for DNA Replication

DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed:

DNA Helicases - These proteins bind to the double stranded DNA and stimulate the separation of the two strands.

DNA single-stranded binding proteins - These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases. Replication is 100 times faster when these proteins are attached to the single-stranded DNA.

DNA Gyrase - This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process.

In addition to these proteins, several other enzymes are involved in bacterial DNA replication.

DNA Polymerase 1 - It was the first enzyme discovered with polymerase activity, and it is the best characterized enzyme. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. That enzyme is DNA Polymerase 3.

There are three activities associated with DNA polymerase 1:

1. 5' to 3' elongation (polymerase activity).
2. 3' to 5' exonuclease (proof-reading activity).
3. 5' to 3' exonuclease (repair activity).

The second two activities of DNA Polymerase 1 are important for replication, but it is the DNA Polymerase 3 enzyme that performs the 5'-3' polymerase function.

Primase - The requirement for a free 3' hydroxyl group is fulfilled by the RNA primers that are synthesized at the initiation sites by these enzymes.

DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups.

Synthesis of DNA

When DNA is synthesized, the free 3´ hydroxyl (OH) group from the growing strand of DNA attacks the phosphate on the next base to be added. Pyrophosphate is released and the new base forms a phosphodiester bond with the growing strand of DNA. The free 3´ hydroxyl group is then freed to attack the next base to be added. This reaction is catalyzed by DNA Polymerase. Similar reactions are catalyzed by RNA Polymerasein during the synthesis of RNA.

Because the strands of a DNA double helix have opposite chemical polarity, one strand is extended in a 5'-3' direction while the other extends in a 3'-5' direction. However, DNA polymerases can only catalyze synthesis in the 5'-3' direction.

Thus, the 5'-3' strand (known as the Leading strand), has continuouse synthesis. The other, in the 3'-5' direction, (known as the lagging strand), has discontinuous synthesis. (refer to diagram on the left.) Thus, the lagging strand synthesizes in small, short fragments known as Okazaki fragments (so named after Reiji & Tuneko Okazaki, the founders).

The short fragments synthesize in a 5'-3' direction, giving rise to the discontinuous break in the synthesis.

Next, the DNA primase synthesizes the RNA primers at the initiation site (see diagram below.) Following that, DNA polymerase 3 will extend off the RNA primers. DNA polymerase 1 has both a removal and a synthesizing activity . As such, simultaneously, the RNA primers are removed by 5'-3' exonuclease activity of DNA polymerase 1, and also carrying out synthesis by the polymerase activity of DNA polymerase 1 at the same time.

Finally, the DNA chain is covalently closed by the DNA ligase.